Cereal-based food product co-fermented with propionibacterium

Abstract

The present invention relates to fermented food products, in particular low-alcohol food products, preferably a beverage or condiment, prepared using cereal malt wort. These fermented products are obtained by co-fermenting the wort with one or more propionibacteria and one or more yeast(s) for alcohol fermentation.

Description

[0001] Cereal-based food product co-fermented with PropionibacteriumFIELD OF THE INVENTION The present invention belongs to the field of fermented food products, preferably a beverage or condiment, prepared from cereal malt wort, in particular low-alcohol food products. These fermented products are obtained by co-fermenting the wort with one or more propionic bacteria and one or more alcoholic fermentation yeasts. STATE OF THE ARTThe market for fermented food products, and in particular low-alcohol beverages, is continuously growing. The production of low-alcohol beverages has so far been carried out by physical or biological processes. Existing biological processes mainly involve the use of low-attenuation yeasts, lactic acid bacteria and acetic acid bacteria.However, there remains a need for low-alcohol food products prepared from cereal malt wort with interesting organoleptic characteristics such as persistent taste in the mouth, the presence of fruity aromas or balanced acidity. Such products can be obtained by co-fermentation of a cereal malt wort with one or more propionic acid bacteria and one or more alcoholic fermentation yeasts. Propionic acid bacteria are bacteria of the genus Propionibacterium. These bacteria are capable of producing propanoic acid, also called propionic acid, during fermentation. These bacteria can be isolated from dairy products, among others. The bacterium Propionibacterium freudenreichii is a Gram-positive, non-spore-forming, anaerobic and aero-tolerant anaerobic or facultative anaerobic bacterium.It is isolated from milk and dairy products and is used in the food industry as a probiotic and for the production of vitamin B12. 1This bacterium was able to be used in a study of the properties of rice-based beverages fermented with lactic acid bacteria and Propionibacterium.2 In this study, Propionibacterium freudenreichii was cultivated alone or in co-culture with lactic acid bacteria, bifidobacteria and streptococci in a medium containing rice. Fermentation with Propionibacterium alone was insufficient to obtain a product with an acidity level similar to that of milk-based yogurt. Its co-culture with lactic acid bacteria makes it possible to produce a rice-based beverage in order to substitute milk of animal origin. The Applicant surprisingly discovered that the co-culture of propionic bacteria with alcoholic fermentation yeasts made it possible to obtain low-alcohol food products based on cereal malt wort which nevertheless retain interesting organoleptic characteristics. DISCLOSURE OF THE INVENTION Continuing itsefforts, the Applicant has developed a process for preparing a fermented cereal-based food product, preferably a drink or condiment, which is not or only slightly alcoholic. Thus, a first subject of the invention is a process for preparing a fermented cereal-based food product, preferably a beverage or condiment, with little or no alcohol, comprising the following steps:i) inoculation of a cereal malt wort with one or more propionic bacteria and one or more alcoholic fermentation yeasts, simultaneously or sequentially;ii) co-cultivation of the malt wort for 1 day to 3 months, preferably 3 days to 5 weeks, more preferably 2 to 4 weeks;characterized in that the propionic bacteria and the alcoholic fermentation yeast are inoculated in a yeast:bacteria ratio making it possible to obtain a propionic acid concentration of at least 1.5 g / L, preferably at least 2 g / L, and aalcohol content less than 3%, preferably less than 2%, preferably less than 0.5%.A second subject of the invention is a fermented cereal-based food product with little or no alcohol that can be obtained by the process according to the invention. DESCRIPTION OF THE FIGURESFigure 1A: Glucose and organic acid concentrations as a function of fermentation time in the absence of hops in barley malt wort for three conditions: anaerobiosis 30°C; aerobiosis 30°C; aerobiosis 20°CFigure 1B: Glucose and organic acid concentrations as a function of fermentation time in the presence of hops in barley malt wort for three conditions: anaerobiosis 30°C; aerobiosis 30°C; aerobiosis 20 °CFigure 2A: Concentrations of glucose, glycerol and organic acids as a function of fermentation time in the absence of hops in barley malt wort under aerobiosis 20 °CFigure 2B: Concentrations of glucose, glycerol and organic acids infunction of fermentation time in the presence of hops in barley malt wortFigure 3A: Maltose, glycerol and ethanol concentrations as a function of fermentation time in the absence of hops in barley malt wortFigure 3B: Glycerol and organic acid concentrations as a function of fermentation time in the absence of hops in barley malt wortFigure 4A: Maltose, glucose, glycerol and ethanol concentrations as a function of fermentation time in the absence of hops in barley malt wort – scaling upFigure 4B: Glycerol and organic acid concentrations as a function of fermentation time in the absence of hops in barley malt wort – scaling upFigure 5A: Maltose and ethanol concentrations as a function of fermentation time in wheat malt wortFigure 5B: Acid concentrations organic and glycerol as a function of fermentation time in wheat malt wortFigure 6A: Maltose and ethanol concentrations infunction of fermentation time in rye malt wortFigure 6B: Concentrations of organic acids and glycerol as a function of fermentation time in rye malt wortFigure 7A: Concentrations of maltose and ethanol as a function of fermentation time in barley malt wort with hops (IBU 7)Figure 7B: Concentrations of organic acids and glycerol as a function of fermentation time in barley malt wort with hops (IBU 7)Figure 8A: Concentrations of maltose and ethanol as a function of fermentation time in barley malt wort with hops (IBU 20)Figure 8B: Concentrations of organic acids and glycerol as a function of fermentation time in barley malt wort with hops (IBU 20) DEFINITIONSFor the purposes of the present invention, "no or little "alcoholic" means that the food product has a low alcohol content, i.e., it contains less than 3.0% by volume of ethanol relative to the total volume of the food product. The term"Alcohol content" refers to the % by volume of ethanol in the food product. More specifically, an alcohol-free or non-alcoholic food product corresponds to a product with an alcohol content of less than 0.5% (v / v). Also for the purposes of the present invention, a low-alcohol product corresponds to an alcohol content of more than 0.5% (v / v) and less than 3% (v / v). In the context of the present invention, the terms "co-culture" and "co-fermentation" are used identically. For the purposes of the present invention, a range of values ​​designated by the expression "between a and b" represents the range of values ​​from strictly greater than a to strictly less than b (i.e. excluding the limits a and b), while any range of values ​​designated by the expression "from a to b" represents the range of values ​​from a to b, i.e. including the strict limits a and b. For the purposes of the present invention, the term "a" or "an" means "one orseveral" or "at least one".DETAILED DESCRIPTION OF THE INVENTION The method according to the invention is a method for preparing a fermented cereal-based food product, preferably a beverage or condiment, with little or no alcohol, comprising the following steps:i) inoculation of a cereal malt wort with one or more propionic bacteria and one or more alcoholic fermentation yeasts, simultaneously or sequentially;ii) co-cultivation of the malt wort for 1 day to 3 months, preferably 3 days to 5 weeks, more preferably 2 to 4 weeks;characterized in that the propionic bacteria and the alcoholic fermentation yeast are inoculated in a yeast:bacteria ratio making it possible to obtain a propionic acid concentration of at least 1.5 g / L, preferably at least 2 g / L and an alcohol content of less than 3%, preferably less than 2%, preferably less than 0.5%. Cereal malt wort,propionic bacteria and alcoholic fermentation yeasts Wort is a mixture obtained by pressing or cooking plants or plant extracts. In the context of the present invention, the wort is obtained from cereal malt. Malt corresponds to cereal grains that are germinated under conditions well known to those skilled in the art. The malt is mainly intended to undergo further fermentation. According to a preferred embodiment, the cereal malt wort is selected from the list consisting of barley malt wort, wheat malt wort, rye malt wort, sorghum malt wort, buckwheat malt wort, spelt malt wort, oat malt wort, rice malt wort, corn malt wort, millet malt wort and mixtures thereof, preferably barley malt wort and mixtures thereof with wheat malt wort, rye malt wort, sorghum malt wort, buckwheat malt wort,spelt, oat malt wort, rice malt wort, corn malt wort, millet malt wort, more preferably barley malt wort. The wort will preferably be chosen according to its capacity to generate at least 1.5 g / L of propionic acid in the presence of the bacteria alone under standard conditions. In the context of the present invention, the cereal malt wort may also include hops and / or other aromatics such as spices or honey. Propionic bacteria are bacteria capable of producing propionic acid in large quantities during fermentation. The propionic acid bacterium(s) may be chosen from the genus Propionibacterium, preferably from the group consisting of Propionibacterium freudenreichii, Propionibacterium thoenii, Propionibacterium jensenii and Propionibacterium acidipropionici, preferentially Propionibacterium freudenreichii. Particularly preferably, the propionic acid bacteriumor one of the propionic bacteria is the strain Propionibacterium freudenreichii subsp. shermanii DSMZ number 20270. Alcoholic fermentation yeasts are yeasts that convert sugars into ethanol. Preferably, the alcoholic fermentation yeast(s) is / are selected from the genus Saccharomyces, preferably from the group consisting of Saccharomyces bayanus, Saccharomyces beticus, Saccharomyces fermentati, Saccharomyces paradoxus, Saccharomyces pastorianus, Saccharomyces uvarum and Saccharomycescerevisiae, more preferably Saccharomyces cerevisiae and Saccharomycespastorianus. Step i) According to step i) of the process of the invention, a cereal malt wort is inoculated with one or more propionic bacteria or several alcoholic fermentation yeasts. This inoculation can be carried out simultaneously or sequentially by techniques known to those skilled in the art. In other words, the cereal malt wort can beinoculated concomitantly with the bacteria and yeasts, it can also be inoculated initially with the propionic bacteria and then the alcoholic fermentation yeast or vice versa. If the inoculation is carried out with several different bacteria and / or yeasts, the inoculation can be carried out alternately with bacteria and yeasts. When the inoculation is carried out sequentially, each inoculation can be carried out from 1 hour to 14 days apart from the following inoculation. According to any of the variants of the invention, the inoculation is carried out in a container or reactor. The following steps can take place in the same reactor. A person skilled in the art will be able to carry out the inoculation using known techniques. The propionic bacteria(s) and the alcoholic fermentation yeast(s) are inoculated in a yeast:bacteria ratio which makes it possible to obtain a propionic acid concentration of at least 1.5 g / L, preferably at least 2 g / Land an alcohol content of less than 3%, preferably less than 2%, preferably less than 0.5%. The quantities of propionic bacteria and alcoholic fermentation yeasts are not limited according to the invention. The bacteria generate propionic acid which limits the activity of the alcoholic fermentation yeast. A person skilled in the art is able to determine the quantity necessary to regulate the alcohol content and the propionic acid concentration in the food product. In particular, these quantities may depend on the cereal or mixture of cereals used to prepare the food product. Typically, the yeast:bacteria ratio in cells / mL is between 0.003 and 2, preferably between 0.005 and 1. The yeast:bacteria ratio in cells / mL may be between 0.5 and 1, preferably be equal to 0.8. The yeast:bacteria ratio in cells / mL can also be between 0.05 and 0.5, preferably equal to 0.08. This ratiocan also be between 0.005 and 0.05, preferably be equal to 0.008. The propionic bacteria can be inoculated into the cereal malt wort at a concentration between 1.00*10 5 and 1.00*10 10 , preferably between 1.00*10 6 and 1.00*10 8cells / mL. Step ii) According to step ii) of the process of the invention, the malt wort is co-cultured for 1 day to 3 months, preferably 3 days to 5 weeks, more preferably 2 to 4 weeks. It is understood that the starting point of the co-culture duration is when at least one propionic bacteria and at least one alcoholic fermentation yeast have been inoculated into the cereal malt wort. This step can be carried out under anaerobic or tolerant aerobic conditions, preferably protected from light. According to a preferred embodiment, this step is carried out at a temperature between 12 and 35 °C, preferably between 20 and 30 °C, more preferably between 20 and 25 °C. At this temperature, the yeast will consume the remaining oxygen in the wort container and then start fermentation.Other steps and end of processThe cereal malt wort can be prepared according to methods known to those skilled in the art.For example, the wort can be obtained from cereal malt by crushing and then mashing. Prior to step i) of the process according to the invention, the cereal malt wort can be heated to a temperature of between 60°C and 120°C and for a duration of between 15 minutes and 5 hours. According to a particular embodiment, the cereal malt wort does not comprise hops and the process according to the invention does not comprise a step of adding hops. When the process according to the invention aims to produce a food product comprising hops, the latter can be added after step ii) of co-culture and / or added prior to the step of heating the cereal malt wort. According to a variant of the invention, the cereal malt wort can undergo a fermentation other than that with the propionic bacteria and the alcoholic fermentation yeast.In the context of this variant, the method preferably comprises a step a) preceding step i), said step a) being a step of fermentation of the cereal malt wort with a lactic acid bacterium. The lactic acid bacterium is preferably of the genus Lactobacillus. According to another variant of the invention, no other type of bacteria than propionic bacteria is inoculated, in particular no lactic acid bacterium. At the end of these steps of the method according to the invention, the food product can be recovered in a known manner and stored if necessary. The food product is consumable at the end of this process. Optionally, other steps can be carried out within the framework of the present invention. Thus, the method can comprise steps of filtration, maturation of the product and packaging (for example bottling in the context of the preparation of a beverage). These techniques are known to those skilled in the art.It is also possible, according to the invention, to add an additive chosen from flavorings, natural or synthetic sweeteners, gelling agents, pH adjusters, foaming agents and preservatives. Fermented cereal-based food product A second subject of the invention is a fermented cereal-based food product with little or no alcohol that can be obtained by the process according to the invention. This product therefore comprises a propionic acid concentration of at least 1.5 g / L, preferably at least 2 g / L and an alcohol content of less than 3%, preferably less than 2%, preferably less than 0.5%. The food product according to the invention may have a pH < 6, preferably < 5, preferably ranging from 4.5 to 5. The food product according to the invention may comprise at least 10 g / L, preferably at least 20 g / L of maltose.Preferably, the food product according to the invention comprises less than 5 g / L, preferably less than 1 g / L of glycerol. Preferably, the food product according to the invention comprises at least 2.5 g / L, preferably at least 3 g / L of propionic acid. The food product according to the invention may comprise at least 0.5 g / L, preferably at least 1 g / L of acetic acid. According to a particular embodiment, the food product according to the invention comprises between 1 and 3% of alcohol, between 20 and 50 g / L of maltose, between 1.5 and 4 g / L of propionic acid and between 0.5 and 1.5 g / L of acetic acid. According to a particular embodiment, the food product according to the invention comprises between 2.5 and 3% of alcohol, between 20 and 30 g / L of maltose, between 1.5 and 2.5 g / L of propionic acid and between 0.5 and 1.5 g / L of acetic acid.According to another particular embodiment, the food product according to the invention comprises between 1.2 and 1.8% of alcohol, between 40 and 50 g / L of maltose, between 2 and 4 g / L of propionic acid and between 0.5 and 1.5 g / L of acetic acid. According to another particular embodiment, the food product according to the invention comprises less than 1% of alcohol, between 45 and 55 g / L of maltose, between 4.5 and 5.5 g / L of propionic acid and between 1.5 and 2.5 g / L of acetic acid. The concentrations of sugars (maltose, glucose and fructose), ethanol and organic acids (acetic acid, propionic acid) are preferably determined by high performance liquid chromatography (HPLC) according to the method described in the examples. Another subject of the invention relates to a fermented cereal-based food product with little or no alcohol, comprising a propionic acid concentration of at least 1.5 g / L and an alcohol content of less than 3%, preferably less than 0.5%.It may have one or more of the characteristics presented above. Use according to the invention Another subject of the invention relates to the use of a co-culture of one or more propionic bacteria with one or more alcoholic fermentation yeasts in a cereal malt wort to obtain a food product with little or no alcohol. The above-mentioned characteristics of the present invention, as well as others, will be better understood on reading the following description of several examples of embodiments of the invention, given for illustrative and non-limiting purposes. EXAMPLES 1. Materials and methods 1.1 Raw materials and. The malted cereals used for making the mash are barley, wheat and rye. Dumalt 100% Pilsen is used for making the barley malt mash. The alcoholic fermentation yeast lyopSafAle S-04 (Fermentis, Lesaffre) and the bacterium Propionibacterium freudenreichii subsp. shermanii isolated from cheese (DSMZ reference number 20270) are used for the fermentation of the mash. 1.2. Preparation of the culture medium The control culture medium used for the growth of the bacteria contains 10 g / L of peptone, 10 g / L of yeast extract, 2.5 g / L of potassium dihydrogen phosphate, 10 g / L of sodium acetate and 5 g / L of magnesium sulfate. 15 g / L of agar is added to obtain a solid culture medium. The bacteria are cultured and isolated in this medium under optimal culture conditions (30°C anaerobically). Liquid precultures are then created and the cells are stored in a glycerol:water mixture.1.3.Preparation of barley malt wort with and without hops1.3.1. Mashing and filtrationThe malt is crushed using the Ss Brewtech™ crusher to a grind of 2. A classic Ale recipe is used to carry out the tests (top fermentation). Tap water (25 L) is placed in the Brewtools® brewing kettle and preheated to 67 °C. The pH of the water is adjusted to 5.45 with lactic acid (optimal pH for enzyme activities). Mashing is then carried out for 25 min at 64 °C and then for 20 min at 72 °C. During this stage, the enzymes present in the malt are activated and the polysaccharides are therefore hydrolyzed into fermentable sugars. At the end of mashing, the temperature of the wort is increased to 78°C for 5 minutes in order to deactivate the enzymes (“mash out”).1.3.2. RinsingAt the end of the mash out, the filtered wort is transferred to a 2nd Brewtools® tank. The spent grains present in the first tank are rinsed to recover the residual sugars.The water used for rinsing (10 L) is preheated to 77 °C in the Tompress® tank. A volume of wort is taken after rinsing and autoclaved. The resulting medium corresponds to the wort without hops. The plato degree of this wort is equal to 13.1.3.3. Boiling and Whirlpool The remaining wort is brought to a boil for 60 min at 100 °C. The Amarillo hops are put at the beginning of the boil. After 1 hour of boiling, a "whirlpool" is carried out. At the end of this step, a volume of wort is taken and filtered. The resulting medium corresponds to the wort with hops. The plato degree of this wort is equal to 13.1.4. Preparation of wheat and rye malt mashBarley malt or rye malt is crushed using the Ss Brewtech™ crusher to a grind of 2. The malt (1 kg) is placed in a beaker with 4 L of tap water preheated to 67°C. Mashing is then carried out for 25 min at 45°C (optimal temperature for β-glucanase activity) and then for 60 min at 68°C with stirring.During this stage, the enzymes present in the malt are activated and the polysaccharides are therefore hydrolyzed into fermentable sugars. At the end of mashing, the wort obtained is separated from the spent grain by centrifugation. The degreeplato of the wort is adjusted to 13 by adding water. The wort obtained is then autoclaved. Wheat and rye malt worts are more viscous than barley malt wort.1.5. Fermentation in barley malt wort1.5.1. Preliminary testsA first series of tests is launched in 50 mL Falcon tubes in order to understand the metabolism of the bacteria. The control media, wort without hops and wort with hops (40 mL) are inoculated with the bacteria (1x10. 7cells / mL) and placed in an incubator under three conditions: i) at 20°C aerobically; ii) at 30°C aerobically; iii) at 30 °C in anaerobiosis (Fig. 1A and 1B). Co-culture tests (yeast: bacteria ratio = 80) yeast and bacteria are carried out in the wort without hops at 20 °C in aerobiosis. Successive fermentation tests yeast 20 °C (inoculation at 8x108 cells / mL at d = 0) then bacteria 20 °C (inoculation at 1x107 cells / mL after 14 days) and yeast 20 °C then bacteria 30 °C aerobic were also carried out. Samples are taken after 7 days, 21 days and 28 days of fermentation and the supernatants are analyzed by HPLC to determine their composition in sugars (maltose, glucose and fructose), ethanol and organic acids (acetic acid, propionic acid). Aerobiosis means inoculation of the Falcon tubes under normal atmosphere and then closing the tubes without any other precautions.Anaerobiosis means inoculation under normal atmosphere and then the Falcon tubes are closed and placed in a closed container with oxygen absorbers.1.5.2. Validation of preliminary testsFollowing the results of the preliminary tests, the 20 °C aerobic condition was chosen for this second series of tests in order to validate the results obtained previously since aerobic conditions present fewer constraints. The wort without hops and the wort with hops are inoculated with the bacteria (1x10. 7 cells / mL) or yeast (8x10 8cells / mL). Coculture tests (yeast:bacteria ratio = 80) and successive fermentation (yeast then bacteria) were also carried out. Samples were taken after 7, 14, 17, 21, 35 and 51 days and HPLC analyses were performed. The tests were carried out in glass bottles to characterize the aromas obtained as a function of time. 1.5.3. Coculture tests In this third series of tests, the yeast:bacteria ratio was varied (Table 1). Samples were taken after 7, 14, 21 and 28 days of aerobic fermentation at 20 °C. Control fermentation tests with yeast alone (5 different cell concentrations) and with bacteria alone (1x10 7cells / mL) are carried out in order to be able to compare them with the coculture tests. The monitoring of sugars, organic acids and ethanol in the fermentation media was carried out after 7, 14, 21 and 28 days. Cocultures Control Control yeast bacteria Bacteria Yeast Ratio Yeast Bacteria (cells / 7mL) (cells / mL) yeast:bacteria (cell 1 08 es / mL) (cells / mL) 1x10 8x 80 8x108 1x1071x107 8x107 8 8x107 1x1071x107 8x106 0.8 8x106 1x1071x107 8x105 0.08 8x105 1x1071x107 8x104 0.008 8x104 1x107Table 1: Concentrations of bacteria and yeast cells for control trials and coculture trials 1.5.4. Scale-upFollowing the co-culture trials in Falcons, the yeast:bacteria ratios 0.8, 0.08 and 0.008 were selected to verify the possibility of scaling up. The barley malt wort without hops (3.5 L) is then placed in a bucket and then inoculated with the yeast (8x10 8 cells / mL) or bacteria (8x10 8cells / mL). Coculture tests with the 3 selected ratios (ratios 0.8, 0.08 and 0.008: see Table 1) were carried out. An additional ratio (0.0008) was tested in the buckets. The buckets are placed at 20 °C and samples are taken after 7 and 28 days to analyze the composition of the media by HPLC. 1.6. Fermentation in wheat and rye malt worts The objective of this series of tests is to verify the possibility of producing low-alcohol beverages using other types of malted cereals. Rye or wheat malt worts are therefore used. Coculture tests are carried out with 5 yeast:bacteria ratios (see Table 1). Control fermentation tests with yeast alone (5 different cell concentrations) and with bacteria alone (1x107 cells / mL) are carried out in order to be able to compare them with the coculture tests.Sugars, organic acids and ethanol in the fermentation media were monitored after 7, 14 and 21 days. The decision not to continue fermentation up to 28 days was made following the results obtained in the barley malt wort (no difference in the composition of the medium between 21 and 28 days). 1.7. Fermentation in a barley malt wort with different quantities of hops The objective of this series of tests is to verify that the bacteria grows in the presence of hops in order to produce "low alcohol" and alcohol-free beer. The bacterium Propionibacterium freudenreichii was subcultured several times in a wort containing increasing concentrations of hops under its optimal culture conditions (30 °C anaerobically). Liquid precultures are then made and the cells adapted to the presence of hops are stored in a glycerol:water mixture. The preparation of the wort is described above.Magnum hops (containing 13.3% α-acids): 0.38 g / L (IBU 7) and 0.77 g / L (IBU 20) are added during the boiling step. Barley malt worts (IBU 7 and IBU 20) are then inoculated with the bacteria or yeast. Co-culture tests are carried out with 3 yeast:bacteria ratios (0.8; 0.08 and 0.008). 1.8. Method for determining concentrations by HPLC The concentrations of D-Glucose, D-Fructose, D-Maltose, Glycerol, organic acids and ethanol are analyzed by HPLC (Alliance). The filtered and diluted samples (10 μL) are passed through a 150mm*7.8mm ion exclusion column. The column temperature is 45 °C. Sulfuric acid (0.005 N) is used as the mobile phase at a flow rate of 1 mL / min. The refractive index (RI) detector is used for the analysis of sugars, ethanol, and glycerol. The UV detector is used for the analysis of organic acids. 2. Results 2.1. Barley malt wort 2.1.1.Preliminary tests The hop-free wort medium initially contains 44 g / L of maltose and 9 g / L of glucose (absence of organic acids). Figure 1A shows the glucose and organic acid concentrations as a function of fermentation time in the hop-free wort in the presence of Propionibacterium freudenreichii and for the three conditions tested: anaerobiosis 30°C, aerobiosis 30°C and aerobiosis 20°C. Maltose is not shown because its concentration did not vary over time. It is therefore not consumed by the bacteria. It is noted that glucose is completely consumed after 7 days at 30°C (optimal growth temperature of the bacteria) while its consumption is slower at 20°C. The propionic acid concentration is approximately 4.8 g / L after 21 days for the 3 conditions tested. When all the glucose is consumed, the acetic acid produced by the bacteria is consumed after 14 days.Table 2 shows the concentrations of sugars, organic acids, glycerol and ethanol for the coculture tests (yeast:bacteria ratio = 80). In the presence of yeast, the amount of propionic acid produced by the bacteria (1.6 g / L: see Table 2) after 21 days is lower than that produced by the bacteria when it is alone in the medium (4.8 g / L: see Figure 1A). In coculture with the bacteria, the yeast produces 24 g / L of ethanol (Table 2). In the absence of the bacteria, 41 g / L of ethanol is obtained after 28 days of fermentation for the yeast alone inoculated at 8x108 cells / mL (20°C) (see Figure 4B). The coculture tests also showed that the bacteria consumes the glycerol produced by the yeast (see Table 2). All the maltose is consumed by the yeast after 7 days. Day Maltose GlucoseAcid Propionic acetic acidGlycerol Ethanol0 43.8 9.3 0.0 0.0 0.0 0.07 0.0 0.0 0.7 0.9 4.2 24.514 0.0 0.0 0.0 1.4 3.0 24.321 0.0 0.0 1.1 1.6 0.3 23.4Table 2: Concentrations of sugars, glycerol, ethanol and organic acids (g / L) as a function of fermentation time in the wort without hops (20°C, aerobically) in the presence of yeast (8x108 cells / mL) and bacteria (1x107 cells / mL) (yeast:bacteria ratio = 80) In the wort with hops which initially contains 86 g / L of maltose and 30 g / L of glucose, the glucose concentration varies slightly during fermentation for the 3 conditions tested (Figure 1B). Acetic acid was not detected in the samples taken, its concentration is below the detection limit. The propionic acid concentration decreases from 2.4 g / L (at 14 days) to 0 g / L (at 21 days) for the 3 conditions tested: anaerobiosis 30°C, aerobiosis 30°C and aerobiosis 20°C. The bacteria can consume the propionic acid produced by the Wood-Werkman metabolic pathway. 3The metabolism of the bacteria is therefore not the same in the absence and presence of hops in the barley malt wort. 2.1.2. Validation of preliminary tests The wort medium without hops initially contains 77 g / L of maltose and 23 g / L of glucose (absence of organic acids). Figure 2A shows the results of variation of glucose, glycerol and organic acid concentrations as a function of fermentation time at 20 °C in aerobiosis for the bacteria alone, for the yeast alone or for the yeast and bacteria in coculture (yeast: bacteria ratio = 80). The bacteria alone consumes all the glucose then the acetic acid from 35 days. The quantity of propionic acid obtained is 4.7 g / L and maltose is not consumed by the bacteria (not shown). This same trend was observed for the preliminary tests, which confirms the repeatability of the results.In the presence of yeast (alone or in coculture), sugars (glucose and maltose) are consumed quickly (after 7 days) and the yeast produces about 5 g / L of glycerol. For fermentation tests in the presence of hops (Figure 2B), the growth of the bacteria is slowed down and the profile of organic acids and glycerol is slightly different between coculture (yeast:bacteria ratio = 80) and yeast alone. Yeast alone and in coculture produces about 50 g / L of ethanol in the presence of hops (see Table 3). As we observe that ethanol production was not reduced in the presence of bacteria in the wort with hops contrary to the results of the preliminary tests, a third series of tests was launched with variation of the yeast:bacteria ratio. Yeast (8x108 cells / mL) Coculture (yeast:bacteria ratio = 80) J. 7 52.4 55.7J14 51.5 48.2J21 50.2 52.3J51 43.9 51.9Table 3: Ethanol concentration (g / L) as a function of fermentation time with yeast alone or in coculture (yeast:bacteria ratio = 80)2.1.3. Co-cultures In this third series of tests, the yeast:bacteria ratio is varied in order to evaluate the production of ethanol and organic acids. The objective is to determine the ratios that allow for alcohol-free or low-alcohol drinks with interesting aromatic profiles. The concentrations of sugars, glycerol, ethanol and organic acids are presented in Figures 3A and 3B for the different ratios and as a function of fermentation time, in comparison with the values ​​for fermentation with bacteria alone. The ethanol concentration decreases from 39 g / L (ratio 80) to 3 g / L (ratio 0.008 which corresponds to 8x10 4cells / mL) and the propionic acid concentration increases from 1.2 g / L (ratio 80) to 4.8 g / L (ratio 0.008) after 28 days of fermentation. In the presence of yeast alone at 8x10 4 cells / mL, all the maltose is consumed and the concentration of ethanol produced is 41 g / L after 28 days of fermentation (41 g / L of ethanol corresponds to an ethanol % of 5.2%: see Table 4). The maltose that is usually consumed by the yeast remains present in the fermentation medium at low yeast:bacteria ratios. This result confirms that at low yeast:bacteria ratios, yeast growth is not inhibited by the lack of substrate but by the presence of propionic acid in the medium. The alcohol percentages obtained by fermentation with yeast alone or in coculture with bacteria in barley malt wort are presented in Table 4. Yeast (cells / mL) Fermentation % alcoholYeast 5.188x10 8Coculture (yeast:bacteria ratio = 80) 4.91 Yeast L evure 4.72 8x10 7 Coculture (yeast:bacteria ratio = 8) 6Yeast 5.218x10 Coculture (ratio = 2.63 5 ,20 yeast: bacteria 4Yeast 5.228x10 Coculture (yeast:bacteria ratio = 0.008) Table 4: % alcohol in fermented barley malt beverages (after 28 days of fermentation) 2.1.4. Scale-up The co-culture results obtained in Falcons showed that the amount of alcohol begins to decrease at a yeast:bacteria ratio equal to 0.8. The ratios ranging from 0.008 to 0.8 were therefore selected to check whether the trend remains the same when increasing the wort volume. The tests are therefore carried out in 4 L buckets and the results of the HPLC analyses are presented in Figures 4A and 4B. The ethanol concentration decreases from 41 g / L (yeast control) to 20, 15 and 9 g / L for the ratios of 0.8, 0.08 and 0.008 respectively. The propionic acid concentration increases from 2 g / L (ratio 0.8) to 4 g / L (ratio 0.008) after 28 days of fermentation. The results are therefore consistent with those obtained in the Falcons. The ratio 0.0008 was tested to have an ethanol % < 1%. 2.2.Wheat malt wort The concentrations of sugars, glycerol, ethanol, and organic acids in wheat malt wort are shown in Figures 5A and 5B for different yeast:bacteria ratios and as a function of fermentation time. The yeast controls shown in the graphs correspond to an initial cell concentration in the medium of 8x10. 8cells / mL (indeed, Table 4 shows that, regardless of the yeast inoculum rate, the final ethanol concentration after 28 days is always the same for fermentation with yeast alone). The ethanol concentration in the wort decreases from 47 g / L (yeast control) to 34 g / L (ratio 0.008) after 21 days of culture (Figure 5A). At the same ratio 0.008, the propionic acid concentration is approximately 4 times lower in wheat malt wort (1.3 g / L) than in barley malt wort (4.7 g / L) for the same culture duration (Figure 5B). The propionic acid concentration in wheat malt wort is therefore not sufficient to inhibit the yeast, which explains the higher ethanol concentrations for low yeast:bacteria ratios. The alcohol percentages obtained by fermentation with yeast alone or in coculture with bacteria in wheat malt wort are presented in Table 5.These results demonstrate that the concept of yeast:bacteria coculture to obtain a reduced alcohol content works for all types of cereals: when the yeast:bacteria ratio decreases, the alcohol content decreases. A person skilled in the art will know how to optimize the conditions, in particular the yeast:bacteria ratio and the quantity of yeast or bacteria introduced, to obtain the desired alcohol contents. Yeast (cells / mL) Fermentation % alcohol Yeast 5.928x10. 8 Coculture (yeast:bacteria ratio = 80) L evure 5,99 8x10 7 Coculture (yeast:bacteria ratio = 8) L evure 6,05 8x10 6 Coculture (yeast:bacteria ratio = 0.8) L evure 5,97 8x10 5 Coculture (yeast:bacteria ratio = 0.08) 5.24 4Yeast 5.898x10 Coculture (yeast:bacteria ratio = 0.008) 4.33Table 5: % alcohol in fermented wheat malt beverages (after 21 days of fermentation) 2.3. Malt wort In rye malt wort, the ethanol concentration decreased from 44 g / L (yeast control: inoculum rate 8x108 cells / mL) to 29.8 g / L (ratio 0.008) after 21 days of coculture fermentation for different yeast:bacteria ratios (Figure 6A). The bacteria alone produced less propionic acid in rye malt wort (3 g / L) than in barley malt wort (Figure 6B). Consequently, coculture of bacteria with yeast in rye malt wort resulted in higher amounts of ethanol and lower amounts of propionic acid compared with barley malt wort. The alcohol percentages obtained by fermentation after 21 days with yeast alone or coculture with bacteria in rye malt wort are presented in Table 6. It is observed that in rye malt wort too, when the yeast:bacteria ratio decreases, the alcohol content decreases.The skilled person will know how to optimize the conditions, in particular the yeast:bacteria ratio and the quantity of yeast or bacteria introduced, to obtain the desired alcohol contents. Yeast (cells / mL) Fermentation % alcohol 8x10. 8Yeast 5.14 Coculture (ratio 5.35 7 yeast:bacteria = 80) 8x10 Yeast 5.39 Coculture (ratio 5.46 yeast:bacteria = 8) 8x106 Yeast 5.61 Coculture (ratio 5.23 5 yeast:bacteria = 0.8) 8x10 Yeast 5.19 Coculture (ratio 5.04 4 yeast:bacteria = 0.08) 8x10 Yeast 5.49 Coculture (ratio 3.77 yeast:bacteria = 0.008) Table 6: % alcohol in fermented beverages based on rye malt (after 21 days of fermentation) 2.4. Fermentation in barley malt wort with different amounts of hops The concentrations of maltose, glycerol, ethanol and organic acids in barley malt worts with hops are shown in Figures 7A, 7B, 8A and 8B for the different coculture ratios and as a function of fermentation time.The ethanol concentration decreases from 41 g / L (yeast control) to 33 g / L (ratio 0.8 which corresponds to 8x106 cells / mL) and the propionic acid concentration increases from 0.7 g / L (yeast control) to 1.6 g / L (ratio 0.8) in the wort with hops (IBU 7) after 21 days of fermentation. For this same ratio, the amount of ethanol is 24 g / L and the amount of propionic acid is 2 g / L in a wort without hops. For the ratios 0.08 and 0.008, the amounts of ethanol and propionic acid are identical in the wort without or with hops (IBU 7). In the wort with hops (IBU 20), the ethanol concentration decreases from 41 g / L (control and ratio 0.8) to 27 g / L (ratio 0.08) and 10.9 g / L (ratio 0.008) (Figure 8). Increasing the quantity of hops therefore slows down the development of the bacteria but does not prevent the production of alcohol contents below 1.5%, regardless of the quantities of hops (see Table 7). Fermentation % alcoholWithout hops IBU 7 IBU 20Bacteria (1x10.7 cells / mL) 0 0 0 Yeast (8x10 8 cells / mL)5.2 5.2 5.2Coculture (yeast:bacteria ratio = 0.8) 3 4.2 5.2Coculture (yeast:bacteria ratio = 0.08) 1.5 1.6 3.4Coculture (yeast:bacteria ratio = 0.008) 0.4 0.6 1.4Table 7: % alcohol in fermented barley malt beverages in the absence and presence of hops (IBU 7 and 20) (after 21 days of fermentation)

[0002] BIBLIOGRAPHICAL REFERENCES1. Chamlagain et al. 2018. In situ production of active vitamin B12 in cereal matrices using Propionibacterium freudenreichii. Food Science & Nutrition, 6(1), 67.2. Cichońska et al. 2022. Properties of rice-based beverages fermented with lactic acid bacteria and Propionibacterium. Molecules, 27(8), 2558.3. Tangyu et al. 2022. Co-cultures of Propionibacterium freudenreichii and Bacillusamyloliquefaciens cooperatively upgrade sunflower seed milk to high levels of vitamin B12 and multiple co-benefits. Microbial cell factories, 21(1), 1.

Claims

CLAIMS 1. Procédé de préparation d’un produit alimentaire fermenté à base de céréales, de preferably a drink or condiment, not or little alcoholic including the steps suivantes : i) inoculation d’un moût de malt de céréales avec une ou plusieurs bactérie(s) propionique(s) and one or more alcoholic fermentation yeast(s), simultaneously or séquentiellement ; ii) co-culture du moût de malt pendant 1 jour à 3 mois, de préférence 3 jours à 5 semaines, de préférence encore de 2 à 4 semaines ; characterized in that the propionic bacteria and the alcoholic fermentation yeast are inoculated in a yeast:bacteria ratio making it possible to obtain une concentration en acide propionique d’au moins 1,5 g / L, de préférence d’au moins 2 g / L et une teneur en alcool inférieure à 3 %, de préférence inférieure à 2 %, préférentiellement inférieure à 0,5 %.

2. Method according to claim 1, characterized in that the propionic bacteria(s) est / sont choisie(s) dans le genre Propionibacterium, de préférence dans le groupe constitué de Propionibacterium freudenreichii, Propionibacterium thoenii, Propionibacterium jensenii et Propionibacterium acidipropionici, plus préférentiellement Propionibacterium 3. Method according to claim 2, characterized in that the propionic acid bacterium or one of the propionic acid bacteria is the strain Propionibacterium freudenreichii subsp. shermanii DSMZ number 20270.

4. Procédé selon l’une quelconque des revendications 1 à 3, caractérisé en ce que la ou les levure(s) de fermentation alcoolique est / sont choisie(s) dans le genre Saccharomyces, de préférence dans le groupe constitué de Saccharomyces bayanus, Saccharomyces beticus,Saccharomyces fermentati, Saccharomyces paradoxus, Saccharomyces pastorianus, Saccharomyces uvarum and Saccharomyces cerevisiae, more preferably Saccharomyces cerevisiae et Saccharomyces pastorianus.

5. Procédé selon l’une quelconque des revendications 1 à 4, caractérisé en ce que ledit moût de malt de céréales est choisi dans la liste constituée du moût de malt d’orge, du moût de malt de blé, du moût de malt de seigle, du moût de malt de sorgho, du moût de malt de sarrasin, du moût de malt d’épeautre, du moût de malt d’avoine, du moût de malt de riz, du moût de malt de maïs, du moût de malt de millet et leurs mélanges, de préférence du moût de malt d’orge et ses mélanges avec le moût de malt de blé, le moût de malt de seigle, le moût de malt de sorgho, le moût de malt de sarrazin, le moût de malt d’épeautre, le moût de malt d’avoine, le moût de malt de riz, le moût de malt de maïs, le moût de malt millet, more preferably barley malt wort.

6. Method according to any one of claims 1 to 5, characterized in that the step ii) de co-culture est réalisée en conditions anaérobie ou aérobie tolérante, de préférence à protected from light.

7. Method according to any one of claims 1 to 6, characterized in that the step ii) de co-culture est réalisée à une température comprise entre 12 et 35 °C, de préférence entre 20 et 30°C, de préférence encore entre 20 et 25 °C.

8. Method according to any one of claims 1 to 7, characterized in that the yeast:bacteria ratio in cells / mL is between 0.003 and 2, preferably is between 0.005 and 1.

9. Method according to any one of claims 1 to 8, characterized in that the propionic bacteria is / are inoculated into the cereal malt wort at a concentration comprise entre 1,00*105 et 1,00*1010, de préférence entre 1,00*106 et 1,00*108cells / mL.

10. Method according to any one of claims 1 to 9, characterized in that the must de malt de céréales comprend en outre du houblon.

11. Method according to any one of claims 1 to 10, characterized in that it comprises a step a) preceding step i), said step a) being a step of fermentation of the cereal malt must with a lactic acid bacterium.

12. Produit alimentaire fermenté à base de céréales pas ou peu alcoolisé susceptible d’être obtained by the method according to any one of the preceding claims.

13. Produit alimentaire fermenté à base de céréales selon la revendication 12, présentant un pH < 6, de préférence < 5, préférentiellement allant de 4,5 à 5.

14. Produit alimentaire fermenté à base de céréales selon la revendication 12 ou 13, comprising at least 10 g / L, preferably at least 20 g / L of maltose.

15. Produit alimentaire fermenté à base de céréales selon l’une quelconque des revendications 12 à 14, comprenant moins de 5 g / L, de préférence moins de 1 g / L de glycerol.

16. Produit alimentaire fermenté à base de céréales selon l’une quelconque des revendications 12 à 15, comprenant au moins 2,5 g / L, de préférence au moins 3 g / L d’acide propionic.

17. Produit alimentaire fermenté à base de céréales selon l’une quelconque des revendications 12 à 16, comprenant au moins 0,5 g / L, de préférence au moins 1 g / L d’acide acetic.

18. Produit alimentaire fermenté à base de céréales selon l’une quelconque des revendications 12 à 15, comprenant entre 1 et 3 % d’alcool, entre 20 et 50 g / L de maltose, entre 1,5 et 4 g / L d’acide propionique et entre 0,5 et 1,5 g / L d’acide acétique.

19. Produit alimentaire fermenté à base de céréales selon l’une quelconque des revendications 12 à 17, comprenant moins de 1 % d’alcool, entre 45 et 55 g / L de maltose, between 4.5 and 5.5 g / L of propionic acid and between 1.5 and 2.5 g / L of acetic acid.

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