MALT SPROUTS EXTRACTS AND THEIR USES

Abstract

Disclosed are compositions containing malt sprouts fraction extracts, their process of preparation and their uses.

Claims

1-29. (canceled)

30. Composition containing malt sprouts fraction extract, wherein the composition comprises at least 0.5%, and less than 98% of dry matter by total weight of the composition, and comprising: total Nitrogen content from 40 to 140 g/kg of dry matter free Amino Nitrogen content from 6 to 60 g/kg of dry matter, and nucleic Acid Derivatives such as purines and pyrimidines bases in an amount from 3 to 25 g/kg of dry matter, PUPY content from 1 to 20 g/kg of dry matter, ratio PUPY/Nucleic Acid Derivatives higher than 20%.

31. Composition according to claim 30, consisting of malt sprouts fraction extract wherein the malt sprouts fraction extract comprises at least 0.5%, and less than 98% of dry matter by total weight of the malt sprouts fraction extract and comprising: total Nitrogen content from 40 to 80 g/kg of dry matter free Amino Nitrogen content from 6 to 30 g/kg of dry matter, and nucleic Acid Derivatives such as purines and pyrimidines bases in an amount from 4 to 25 g/kg of dry matter, PUPY content from 3 to 20 g/kg of dry matter, ratio PUPY/Nucleic Acid Derivatives higher than 80%.

32. Composition according to claim 30, consisting of malt sprouts fraction extract wherein the extract is in a liquid form comprising at least 0.5%, and less than 1.1%, or at least 1.1%, and less than 3.5%, or at least 3.5%, and less than 5%, or at least 5%, and less than 7% of dry matter by total weight of the malt sprouts fraction extract and comprising: total Nitrogen content from 40 to 80 g/kg of dry matter free Amino Nitrogen content from 6 to 30 g/kg of dry matter, and nucleic Acid Derivatives such as purines and pyrimidines bases in an amount from 5 to 25 g/kg of dry matter, PUPY content from 4 to 20 g/kg of dry matter, ratio PUPY/Nucleic Acid Derivatives higher than 80%.

33. Composition according to claim 30, consisting of malt sprouts fraction extract wherein the extract is in a liquid form, or a concentrated juice, comprising at least 7%, and less than 10%, or at least 10%, and less than 15% of dry matter by total weight of the malt sprouts fraction extract and comprising: total Nitrogen content from 40 to 80 g/kg of dry matter free Amino Nitrogen content from 6 to 30 g/kg of dry matter, and nucleic Acid Derivatives such as purines and pyrimidines bases in an amount from 4 to 25 g/kg of dry matter, PUPY content from 3 to 20 g/kg of dry matter, ratio PUPY/Nucleic Acid Derivatives higher than 80%.

34. Composition according to claim 30, consisting of malt sprouts fraction extract wherein the extract is in a liquid form or a solid form, or a concentrated juice, a slurry or a paste, comprising at least 15%, and less than 60% of dry matter by total weight of the malt sprouts fraction extract and comprising: total Nitrogen content from 40 to 80 g/kg of dry matter free Amino Nitrogen content from 6 to 30 g/kg of dry matter, and nucleic Acid Derivatives such as purines and pyrimidines bases in an amount from 4 to 25 g/kg of dry matter, PUPY content from 3 to 20 g/kg of dry matter, ratio PUPY/Nucleic Acid Derivatives higher than 80%.

35. Composition according to claim 30, consisting of malt sprouts fraction extract wherein the extract is in a solid form, or a paste or a powder, comprising at least 60%, and less than 98% of dry matter by total weight of the malt sprouts fraction extract and comprising: total Nitrogen content from 40 to 80 g/kg of dry matter free Amino Nitrogen content from 6 to 30 g/kg of dry matter, and nucleic Acid Derivatives such as purines and pyrimidines bases in an amount from 4 to 25 g/kg of dry matter, PUPY content from 3 to 20 g/kg of dry matter, ratio PUPY/Nucleic Acid Derivatives higher than 80%.

36. Composition consisting of malt sprouts fraction extract according to of claim 30, wherein the malt is selected from barley, wheat, rye, spelt, corn, millet sorghum, oat, triticale, rice and mixtures thereof, and obtained from an aqueous extraction of malt sprouts fractions obtained by a sieving process and comprising a content of rootlets and acrospires higher than 50%, or higher than 60%, or higher than 65%, or between 65% and 85% by total weight of fractions.

37. Composition consisting of malt sprouts fraction extract, wherein the composition comprises at least 0.5%, and less than 98% of dry matter by total weight of the composition, and comprising: total Nitrogen content from 40 to 140 g/kg of dry matter free Amino Nitrogen content from 6 to 60 g/kg of dry matter, and nucleic Acid Derivatives such as purines and pyrimidines bases in an amount from 3 to 25 g/kg of dry matter, PUPY content from 1 to 20 g/kg of dry matter, ratio PUPY/Nucleic Acid Derivatives higher than 20%, wherein the malt sprouts fraction extract is such as obtained by a method of preparation comprising the following steps: a. dilution of isolated malt sprouts fractions enriched in anyone of rootlets, acrospires, hulk, dust and mixtures thereof in aqueous phase to obtain diluted malt sprouts fractions; b. diffusion of these diluted malt sprouts fractions in aqueous phase followed by a finishing step at pH<6 or >8 and a thermal treatment at 100 C. to obtain two phases, an aqueous one and a solid one; c. separation of the aqueous phase from the solid phase to recover the aqueous phase; d. optionally microfiltration of the aqueous phase on plate filters to remove fine insoluble particles with a lower size than 1 m; e. optionally, treatment of the aqueous phase obtained in step (c) or (d) in order to obtain the desired % of dry matter by total weight of malt sprouts fraction extract; f. and optionally a sterilization step, or, comprising the following steps: a. dilution of isolated malt sprouts fractions enriched in anyone of rootlets, acrospires, hulk, dust and mixtures thereof in aqueous phase to obtain diluted malt sprouts fractions; b. hydrolysis of these diluted malt sprouts fractions in the presence of exogenous enzymes, to obtain two phases, an aqueous one and a solid one; c. separation of the aqueous phase from the solid phase to recover the aqueous phase; d. optionally microfiltration of the aqueous phase on plate filters to remove fine insoluble particles with a lower size than 1 m; e. optionally, treatment of the aqueous phase obtained in step (c) or (d) in order to obtain the desired % of dry matter by total weight of malt sprouts fraction extract; f. and optionally a sterilization step, or, comprising the following steps: a. dilution of isolated malt sprouts fractions enriched in anyone of rootlets, acrospires, hulk, dust and mixtures thereof in aqueous phase to obtain diluted malt sprouts fractions; b. hydrolysis of these diluted malt sprouts fractions in the presence of exogenous enzymes, followed by a finishing step at pH<6 or >8 and thermal treatment at 100 C. to obtain two phases, an aqueous one and a solid one; c. separation of the aqueous phase from the solid phase to recover the aqueous phase; d. optionally microfiltration of the aqueous phase on plate filters to remove fine insoluble particles with a lower size than 1 m; e. optionally, treatment of the aqueous phase obtained in step (c) or (d) in order to obtain the desired % of dry matter by total weight of malt sprouts fraction extract; f. and optionally a sterilization step.

38. Composition consisting of malt sprouts fraction extract according to claim 37, wherein the malt sprouts fractions comprise: dry matter by total weight of the malt sprouts from 92% to 98%, crude protein content from 22.5 to 37 g/100 g of malt sprout fraction free Amino Nitrogen content higher than 200 mg/100 g of dry matter.

39. Composition consisting of malt sprouts fraction extract according to claim 37, wherein the malt sprouts fractions comprise: dry matter by total weight of the malt sprouts from 92% to 98%, crude protein content from 22.5 to 37 g/100 g of malt sprout fraction free Amino Nitrogen content higher than 200 mg/100 g of dry matter, and containing: total fibers content from 43 to 52 g/100 g of dry matter, total carbohydrates content from 7 to 12 g/100 g of dry matter, total sugars content from 4 to 6 g/100 g of dry matter, fats content below 1 g/100 g, ash content from 5 to 7 g/100 g.

40. Composition according to claim 30, further comprising at least one ingredient selected from additional ingredients.

41. Composition according to claim 30, further comprising at least one ingredient selected from protein hydrolysates, organic acids, mineral acids, mineral salts, sugars, polyols and mixtures thereof, or protein hydrolysates originating from soy, rapeseed, pea, alfalfa, wheat protein, casein, gelatin, or meat

42. Composition according to claim 30, further comprising at least one ingredient selected from protein hydrolysates, organic acids, mineral acids, mineral salts, sugars, polyols and mixtures thereof, or protein hydrolysates originating from soy, rapeseed, pea, alfalfa, wheat protein, casein, gelatin, or meat, comprising: total Nitrogen content from 45 to 140 g/kg of dry matter free Amino Nitrogen content from 10 to 60 g/kg of dry matter, and nucleic Acid Derivatives such as purines and pyrimidines bases in an amount from 2 to 10 g/kg of dry matter, PUPY content from 0.5 to 8 g/kg of dry matter, ratio PUPY/Nucleic Acid Derivatives higher than 20%.

43. Composition according to claim 30, further comprising at least one ingredient selected from protein hydrolysates, organic acids, mineral acids, mineral salts, sugars, polyols and mixtures thereof, or protein hydrolysates originating from soy, rapeseed, pea, alfalfa, wheat protein, casein, gelatin, or meat, comprising: total Nitrogen content from 45 to 140 g/kg of dry matter, free Amino Nitrogen content from 10 to 60 g/kg of dry matter, and nucleic Acid Derivatives such as purines and pyrimidines bases in an amount from 2 to 10 g/kg of dry matter, PUPY content from 0.5 to 8 g/kg of dry matter, ratio PUPY/Nucleic Acid Derivatives higher than 20%, said composition being a food ingredient, or a flavor enhancer, used in food for animals or humans, or a culture and/or fermentation medium.

44. Method of preparing a culture and/or performing a fermentation process, comprising a step of adding a composition containing malt sprouts fraction extract, wherein the composition comprises at least 0.5%, and less than 98% of dry matter by total weight of the composition, and comprising: total Nitrogen content from 40 to 140 g/kg of dry matter, free Amino Nitrogen content from 6 to 60 g/kg of dry matter, and nucleic Acid Derivatives such as purines and pyrimidines bases in an amount from 3 to 25 g/kg of dry matter, PUPY content from 1 to 20 g/kg of dry matter, Ratio PUPY/Nucleic Acid Derivatives higher than 20%.

45. Method according to claim 44, wherein the fermentation process comprises adding mycetes including actinomycetes, fungi yeasts and/or bacteriae.

46. Method according to claim 44, for the production of metabolites by said fermentation process.

47. Method according to claim 44, wherein the culture comprises microbial cells, plant cells and/or animal cells.

48. Method according to claim 44, wherein the culture comprises a living micro-organism; and comprising at least a step wherein said composition is added to the culture and/or fermentation medium or replaces it as a substitute.

Description

FIGURES

[0887] FIG. 1: Schematic illustration of malt sprouts, with (1) representing an acrospire (=germ), (2) representing rootlets, (3) representing the husk (=hull), (4) representing a malted barley grain. A malt sprout is constituted of a mix of (1)+(2)+(3)+malt dust.

EXAMPLES

Example 1: Preparation of Malt Sprout Fraction Extracts in Form of Powder (from Simple Diffusion (Powder 1A) and Simple Hydrolysis Processes (Powder 1B))

[0888] The malt sprout fraction used to produce malt sprout fraction extract in this example is characterized in Tables 1 and 2, is ground using a grinder Bauknecht (level 0). The ground fraction is divided into two part of 12 kg to perform extractions in the presence of water in the ratio 1/10 (1 malt fraction/9 water).

TABLE-US-00004 TABLE 1 Physical characterization of malt sprout fraction MSF 1 MSF1 - Physical characterization From crop 2014 Fractions composition (in %) Husk + whole grains 10 Rootlets + Acrospires 72 Dust 18

TABLE-US-00005 TABLE 2 Physico-chemical analysis of MSF1 Units Values Dry u matter % 97.6 Total nitrogen (N) g/100 g 5.1 Total protein (N*6.25) g/100 g 31.7

[0889] The extractions are carried out in a 200 L reactor equipped with a double envelope. One of the slurry is maintained for 2 h at 20 C. at the natural pH of the mixture. The other is maintained for 2 h 30 min at 50 C. and pH 7.5 in the presence of enzymes, then the temperature is raised to 72 C. and maintained at this value until the end of the extraction, a term total of 4 hours. Following a preliminary study, the choice of enzymes fell on a combination of two proteases preparations Sumizyme LP and Protease Plus.

[0890] In both cases, the final mixture is heated at 80 C. for 5 min. Each of the mixtures thus obtained is then subjected to the same protocol of liquid-solid separation comprising: [0891] A first filtration with a filter press Choquenet (P8329 canvas TC) in the presence of diatomaceous earth (DIC/S) under 4 bar pressure, followed by rinsing the filter cake with a volume of osmotic water in a ratio 1/4 of the volume of the first juice and final compaction of the cake to 7.5 bar. [0892] Centrifugation (RC12BP+at 4000 g, 10 min, 20 C.) after the additional filter press was carried out in the second case due to a too large content of residual solid particles. [0893] The juices were then subjected to a second filtration in two steps using a plate filter (KDS15 filters and S80 at 0.7 and 0.2 microns, respectively). [0894] The juices (also named diluted malt sprouts fraction extracts) such obtained are characterized in the following Table 3:

TABLE-US-00006 TABLE 3 Characterization of the diluted malt sprout fraction extracts obtained after plate-filter step. MSFE1-A MSFE1-B Dry matter dm (%) 1.14 3.98 Total nitrogen (g/kg dm) 58.77 65.07 [0895] After plate-filter step the two malt sprout fraction extracts have been concentrated until 8.4% dm and 11.25% dm respectively, before the step of drying on Bchi 190 spray-dryer, at maximum aspiration, feed:200 ml/h (level 4), inlet T C. 117-120 C. (level 4.5-5), outlet T C. 78-82 C. [0896] 2 powders of malt sprout fraction extracts have been recovered following the spray-drying step, characterized in Table 4.

TABLE-US-00007 TABLE 4 Characterization of powdered malt sprout fraction extracts. Powder 1A Powder 1B Dry matter (%) 96.7 97.5 Total nitrogen (g/kg dm) 55.1 63.8 Total FAN (g/kg dm) 14.8 28.7 Total DAN (g/kg dm) 4.0 4.6 Total PUPY (g/kg dm) 3.8 4.4 Ratio PUPY/DAN 97.7 95.5

Example 2: Preparation of Malt Sprout Fraction Extract (from Simple Hydrolysis Process) in the Form of Syrup

[0897] The malt sprout fraction (MSF2) used to produce malt sprout fraction extract in this example is characterized in Tables 5 and 6, is ground using a grinder Bauknecht (level 0). This 14 kg of ground fraction is used to perform extraction in the presence of water in the ratio 1/10.

TABLE-US-00008 TABLE 5 Physical characterization of MSF2 MSF2-Physical characterization From crop 2015 Fractions composition (in %) Husk + whole grains 11 Rootlets + Acrospires 74 Dust 15

TABLE-US-00009 TABLE 6 Physico-chemical analysis of MSF2 Malt fractions blend Units Values Moisture % 6.3 Total nitrogen (N) g/100 g 4.9 Total protein (N*6.25) g/100 g 30.3

[0898] The extraction is carried out in a 200 L reactor equipped with a double envelope. The aqueous extract is adjusted at pH 5 with H.sub.2SO.sub.4 (95%), cellulases and xylanases are added, temperature is maintained for 2 h at 60 C. Then, pH of the aqueous extract is adjusted at 7 and proteases are added, temperature is decreased to 50 C. and maintained at this value until the end of the extraction, a term total of 4 hours.

[0899] At the end of extraction, the final mixture is heated at 80 C. for 5 min. The mixture thus obtained is then subjected to the following protocol of liquid-solid separation comprising: [0900] A first filtration with a filter press Choquenet (P8329 canvas TC) in the presence of diatomaceous earth (DIC/S) under 4 bar pressure, followed by rinsing the filter cake with a volume of osmotic water in a ratio 1/4 of the volume of the first juice and final compaction of the cake to 7.5 bar. [0901] Centrifugation (BTPX centrifuge+at 8000 g, sludge discharge frequency: 30 min, flow 100 liters/hour, 20 C.) after the additional filter press was carried out in the second case due to a too large content of residual solid particles. [0902] The juice were then subjected to a second filtration in two steps using a plate filter (KDS15 filters and S80 at 0.7 and 0.2 microns, respectively). [0903] The malt sprout fraction extract (MSFE2) such obtained is characterized in the following Table 7:

TABLE-US-00010 TABLE 7 Characterization of the MSFE2 obtained after plate-filter step. MSFE2 Dry matter (%) 2.74 Total nitrogen (g/kg dm) 66.05 [0904] After plate-filter step the juice has been concentrated until 60% dm on Heidolph rotovapor (20 liters capacity) at 60 C. [0905] This syrup (slurry concentrate) has been recovered following the concentration step and characterized in Table 8.

TABLE-US-00011 TABLE 8 Characterization of malt sprout fraction extract (MSFE2) in syrup form. MSFE2 (syrup form) Dry matter dm (%) 60.5 Total nitrogen (g/kg dm) 66.06 Total FAN (g/kg dm) 19.05 Total DAN (g/kg dm) 8.02 Total PUPY (g/kg dm) 7.05 Ratio PUPY/DAN (%) 87.9

Example 3: Effect of Malt Sprout Fraction Extract in Powder (Produced from Diffusion Process) in Cell Culture Medium for Yeast

[0906] The malt sprout fraction extract in powder form (powder 1A, whose preparation is detailed in Example 1, and which is characterized in table 4), was used for the culture of Saccharomyces boulardii in liquid medium compared to control medium containing yeast extract (YE 0251PW-L Biospringer).

[0907] Each of the media used contained 1 g N/kg, 10 g of glucose/kg of mineral elements (KH2PO4, MgSO4 7H2O and a solution of trace elements respectively 10, 5.12 and 10 g/kg). The initial pH was adjusted to 5.5; after inoculation with the strain of S. boulardii (SuperSmart Saccharomyces boulardii250 mg) they were incubated at 30 C. in Erlenmeyer stirring at 110 rpm.

[0908] The kinetics were followed to estimate the growth rate in the exponential phase (). Biomass X (yeast dry matter) produced after 24 h and 42 h of fermentation was measured after filtration (0.45) of samples from media, washing the filter cake and then drying at 105 C. for 24 hours.

[0909] The results are presented in the following Table 9:

TABLE-US-00012 TABLE 9 Productions cultures of S boulardii from yeast extract and Malt sprout fraction extract (powder 1A). Media (h.sup.1) X 24 (g/kg) X 42 (g/kg) Control medium Yeast 0.16 8.34 8.09 Extract (YE) Powder 1A (Example 1) 0.18 8.25 10.94
With YE control medium after 24 hours of culture, the biomass concentration is 8.34 g/L then decreased slightly 8.09 g/kg at 42 h culture. This finding is not comparable to results obtained with the powder 1 for which the biomass concentration increases between 24 and 42 h: 10.94 g/kg. At nitrogen iso-concentration (1 g/L to N) and on a sterilized medium after filtration under 0.2, using the same culture conditions, the malt sprout fraction extract (Powder 1A) allows an almost identical growth in a medium composed of yeast extract but lead to a higher biomass production when fermentation continues beyond 24 hours.

Example 4: Effect of Malt Sprout Fraction Extracts in Powder Form (Produced from Simple Hydrolysis Process) in Cell Culture Media

4.1: Preparation of Malt Sprout Fraction Extract in Powder Form (from Simple Hydrolysis Process)

[0910]

TABLE-US-00013 TABLE 10 Physico-chemical analysis of malt sprout fraction used for the example 4 MSF3 Dry matter % 93.7 Total protein (N* 6.25) g/100 g 29.6

[0911] A malt sprout fraction extract in powder form (identified powder 2204) was obtained from the malt sprout fraction MSF3 according to the following protocol: [0912] The malt sprout fraction MSF3 is suspended to 1/10 in water in the presence of cellulolytic enzymes (SD 800) and hemicellulolytic enzymes (GC 220). The initial pH of the mixture is adjusted to 5, its temperature at 60 C. The suspension was then maintained at 60 C. for 105 min and then the temperature is brought to 50 C. over 30 min. Meanwhile, after 2 hours of extraction, the pH is adjusted to 7 and treated with proteases (Plus PIB and Sumizyme LP) in the suspension. The extraction continues 105 minutes at 50 C., then the suspension is heated to 80 C. 5 minutes and cooled down to 50 C. [0913] The suspension is kept 2 hours at that temperature. [0914] The suspension thus obtained is then subjected to the following protocol of liquid-solid separation comprising: [0915] A first liquid-solid separation step, the centrifugation (RC12BP+at 4000 g, 10 min, 20 C.) in order to remove the large solid particles. [0916] The juice obtained from centrifuge was then subjected to a second filtration in two steps using a plate filter (KDS15 filters and S80 at 0.7 and 0.2 microns, respectively). [0917] After plate-filter step the juice has been concentrated until 13% dm, before the step of drying on Bchi 190 spray-dryer, at maximum aspiration, feed: 200 ml/h (level 4), inlet T C. 117-120 C. (level 4.5-5), outlet T C. 78-82 C.

[0918] This provides a malt sprout fraction extract in powder form from MSF3, the powder 2204. Its characterization is detailed in table 11.

TABLE-US-00014 TABLE 11 Characterization of the malt sprout fraction extract in powder form (powder 2204). Powder 2204 Dry matter dm (%) 93.9 Total nitrogen (g/kg dm) 75.9 Total FAN (g/kg dm) 17.2 Total DAN (g/kg dm) 5.9 Total PUPY 5.4 Ratio PUPY/DAN 91.5

4.2. Effect of Malt Sprout Fraction Extract in Powder Form (Produced in Example 4.1.) in Yeast Cell Culture Media

[0919] The powdered malt sprout extractpowder 2204 (Table 11) are used as nitrogen source in yeast culture (Saccharomyces boulardii) liquid medium compared to control medium containing yeast extract (YE 0251PW-L Biospringer) as nitrogen source.

[0920] Each of the media used contained: [0921] 0.55 g N/kg, [0922] 10 g of glucose/kg of mineral elements (KH2PO4, MgSO4 7H2O and a solution of trace elements respectively 10; 5.12 and 10 g/kg). [0923] The initial pH was adjusted to 5.5; after inoculation with the strain of S. boulardii (SuperSmart Saccharomyces boulardii250 mg). [0924] The 2 media (one with powder 2204 and one control with yeast extract) were incubated at 30 C. in Erlenmeyer stirring at 110 rpm.

[0925] The fermentation kinetics were followed to estimate the growth rate in the exponential phase (). Biomass X (yeast dry matter) produced after 24 h and 42 h of fermentation was measured after filtration (0.45) of samples from media, washing the filter cake and then drying at 105 C. for 24 hours. The results are presented in the following Table 12:

TABLE-US-00015 TABLE 12 Productions of cultures of S boulardii using different nitrogen sources: yeast extract and malt sprout fraction extract in powder form in media at iso N concentration of 0.5 g/kg. Initial Total glucose Biomass at 32 h Ratio to Ref nitrogen (S0) max average fermentation X32/S0 the control extracts g/kg g/kg h.sup.1 h.sup.1 (X32) g/kg % X/S Control 119.3 9.3 0.57 0.47 4.73 50.9 1 YE Powder 71.3 9.4 0.61 0.5 5.15 54.8 1.08 2204 = Specific growth rate; X = Biomass; S = glucose concentration; YE = yeast extract.

[0926] These experiments demonstrate that the malt sprout fraction extracts in powder form are excellent sources of nitrogen for the yeast culture leading to growth rates and higher biomass yields than the control medium containing yeast extract.

4.3. Effect of Malt Sprout Fraction Extract in Powder Form in Bacteria Culture Media

[0927] The effect of malt sprout fraction extracts in powder form in bacteria culture media have been measured and evaluated using 2 methods: [0928] Method 1: Culture in flasks with various medium, where each nitrogen source presents in control medium (MRS and M17) such as peptone, beef extract or yeast extract is replaced by malt sprout extract extracts in powder form at the same nitrogen concentration (one by one substitution).

[0929] MRS is a selective culture medium designed to favour the luxuriant growth of Lactobacilli for lab study. Developed in 1960, this medium was named for its inventors (De Man, Rogosa and Sharpe). It contains sodium acetate, which suppresses the growth of many competing bacteria (although some other Lactobacillales, like Leuconostoc and Pediococcus, may grow). This medium has a clear brown colour.

[0930] MRS Typically Contains (Weight/Volume): [0931] 1.0% peptone [0932] 1.0% beef extract [0933] 0.4% yeast extract [0934] 2.0% glucose [0935] 0.5% sodium acetate trihydrate [0936] 0.1% polysorbate 80 (also known as Tween 80) [0937] 0.2% dipotassium hydrogen phosphate [0938] 0.2% triammonium citrate [0939] 0.02% magnesium sulfate heptahydrate [0940] 0.005% manganese sulfate tetrahydrate [0941] pH adjusted to 6.2 at 25 C.

[0942] The yeast and meat extracts and peptone provide sources of carbon, nitrogen and vitamins for general bacterial growth. The yeast extract also contains vitamins and amino acids specifically required by Lactobacilli. Polysorbate 80 is a surfactant which assists in nutrient uptake by Lactobacilli. Magnesium sulfate and manganese sulfate provide cations used in metabolism.

[0943] M17 is a selective media recommended as an improved medium for the growth and enumeration of lactic streptococci and their bacteriophages

[0944] M17 Typically Contains:

[0945] Approximate Formula (per 950 mL): [0946] 5.0 g of Pancreatic Digest of Casein [0947] 5.0 g of Soy Peptone [0948] 5.0 g of Beef Extract [0949] 2.5 g of Yeast Extract [0950] 0.5 g of Ascorbic Acid [0951] 0.25 g of Magnesium Sulfate [0952] 19.0 g of Disodium--glycerophosphate [0953] Method 2: Culture on agar plates with malt sprout fraction extract in powder form as only source of nitrogen (total substitution).

[0954] Medium Preparation: [0955] preparation of a stock solution of each nitrogen sources, [0956] preparation of a stock solution of each salt solutions, [0957] preparation of a stock solution of each sugar solutions, [0958] mix of several elements according to the needs, [0959] pH adjustment to 6.5 for medium MRS and pH to 7.2 for medium M17, [0960] centrifugation to eliminate the turbidity (this turbidity is related to addition of RDM extract), [0961] the media is sterilized by filtration and transferred in flasks previously sterilized by autoclaving.

[0962] Seed Preparation: [0963] the seed culture is grown within the commercial media, [0964] at the end of growth the seeds are centrifuged, the pellets are collected in the physiological water and are centrifuged again, [0965] the pellets are new collected in the physiological water and are distributed in the flasks.

[0966] This protocol avoids the intake of undesired nutrients in the medium. The size of inoculums was defined by previous tests.

[0967] Culture in Flask [0968] The culture conditions are: static growth at 37 C.

[0969] The growing is monitored by: [0970] OD 600 nm, [0971] pH, [0972] [glucose] (YSI) for Lactobacillus, [0973] [lactose] (HPLC) for Lactococcus, [0974] [Organics acid], [ethanol] (HPLC), [0975] Dry Matter after filtration on the final time, [0976] Microbiological enumeration on plat count agar with MRS and M17 media

Culture on Plates Agar

[0977] the culture conditions are: static growth at 37 C. under controlled atmosphere via a candle jar, [0978] three types of medium are tested, [0979] commercial medium MRS or M17, [0980] simulated commercial medium with commercial nitrogen sources, [0981] Malt sprout fraction extract in powder trials (substitution of every other nitrogen sources by MSFE in powder), [0982] for the inoculation, 5 spots of 0.1 l of the different cultures are deposited on the surface of each plates, [0983] the growing of the spot is measured each days during 3 days.

[0984] The malt sprout fraction extract in powder used in these experiments corresponds to the powder 2204 (characterized on table 11).

4.3.1. Lactobacillus Strains

[0985] The cultures of Lactobacillus bacteria in liquid media were studied taking as positive control the medium Man Rogosa Sharpe (MRS) from AES (ref AEB140652). The different sources of nitrogen of the medium MRS are Casein peptones (ref A1402HA), meat extract (ref A17HA) and yeast extract (ref A1202HA) which are available from Biokar.

[0986] These cultures have been prepared according to methods 1 and 2 described above.

[0987] The following strains were used: [0988] Lactobacillus acidophilus (ATCC(=American type culture collection) 4356) [0989] Lactobacillus delbrueckii subsp bulgaricus (ATCC11 842).

[0990] The results are presented in the following Table 13:

TABLE-US-00016 TABLE 13 Results of cultures of two lactobacillus strains in media containing malt sprout fraction extract in powder (powder 2204) AES Powder Powder Powder Control AES Powder 2204 in 2204 in 2204 in (ref Control 2204 substitution substitution substitution Media AEB140652) (recomposed) 100% YE PP EV Powder 0 0 49.79 8.21 22.83 18.75 2204 MS g/L N g/L 3.28 3.59 3.45 3.61 3.52 3.57 Lactobacillus acidophilus (h.sup.1) 0.15 0.2 0.44 0.45 0.47 0.48 10.sup.9 0.13 0.94 1.8 1.8 2.5 2.2 UFC/mL Lactic 12.3 17.8 23 21.7 22.1 21.7 acid g/L Lactobacillus delbrueckii (h.sup.1) 0.032 0.048 0.595 0.394 0.62 0.595 10.sup.8 0.77 0.43 5.75 3.5 6.9 4.5 UFC/mL Lactic 9.6 5.6 8.7 10.9 10.3 9.9 acid g/L Powder 2204 is used in the formulation of AES media recomposed from nitrogen sources from Biokar in substitution of one of these nitrogen sources or all 3 (EFM 100%). YE: yeast extract PP: polypeptones of casein EV: meat extract

4.3.2. Lactococcus and Streptococcus Strains

[0991] The following bacteria were investigated: [0992] Lactoccus lactis subsp lactis (ATCC 11454) [0993] Streptococcus salivarius subsp thermophilus (ATCC19 258).

[0994] Taking as control the M17 medium Sigma Aldrich (ref. 56156 M17 broth). Tryptone (ref A1401HA), meat peptones (ref A1708HA) and soy (ref A1601HA), meat extract (ref A170HA) and yeast extract (ref A1202HA) are available from Biokar. The lactose concentration was increased to 20 g/L. The culture conditions and the measurements are similar to that of the preceding example. The results are presented in the following Table 14:

TABLE-US-00017 TABLE 14 Results of cultures of two strains lactococcus and streptococcus in media containing malt sprout fraction extract in powder. Powder Powder Powder Powder Powder SIGMA SIGMA Powder 2204 in 2204 in 2204 in 2204 in 2204 in Commercial Recomposed 2204 substitution substitution substitution substitution substitution Media Control Control 100% YE PS EV T PV Powder 0 0 31.04 4.11 7.65 9.37 4.81 5.11 2204 Dry Matter g/L N g/L 2.0 2.09 2.05 2.1 2.05 2.1 2.1 2.06 Lactococcus lactis (h.sup.1) 1.2 0.71 1.26 1 1.18 1.23 0.822 1.09 Lactic 8.31 7.5 9.2 8.7 8.8 8.9 8.69 8.7 acid g/L Streptococcus salivarius (h.sup.1) 1.03 0.9 0.99 0.73 0.95 1.11 0.88 0.97 10.sup.9 nd 1.1 nd 1.5 4.4 2.4 2.9 7.1 UFC/mL Lactic 7.57 8.35 8.56 8.58 8.71 8.8 8.55 8.7 acid g/L Powder 2204 is used in the formulation media SIGMA recomposed from nitrogen sources from Biokar in substitution of one of these nitrogen sources or all 5 (EFM 100%). YE: yeast extract PS: Soy peptone EV: meat extract PV: Meat peptone T: Tryptone (*): Counts higher than 10.sup.10

[0995] We further proceed to the culture of four strains disclosed in examples 4.3.1 and 4.3.2 in solid agar media.

[0996] These cultures were performed on agar plates, static at 37 C. under controlled atmosphere Candle jars. Three types of media were used: [0997] MRS or M17 media (same references than in examples 4.3.1 and 4.3.2), [0998] MRS and M17 media recomposed from commercial sources of nitrogen, [0999] media consisting of malt sprout fraction extract in powder (PMSFE) in substitution of the total nitrogen sources
5 drops of 0.1 l of different cultures are deposited on the surface of each agar media box. Growth is measured each day for 3 days.

[1000] The results are presented in the following Table 15:

TABLE-US-00018 TABLE 15 Culture of lactic acid bacteria strains in agar media - comparison of colony diameters (in millimeters mm). 24 h 48 h 72 h L acidophilus MRS 5.6 6.1 6.4 MRS recomposed 6.3 6.8 7.3 PMSFE medium 7.3 8.1 8.4 L delbrueckii MRS 5.2 6.6 7.3 MRS recomposed 7.3 8 9.1 PMSFE medium 7.2 8.3 8.7 Lactococcus lactis M17 4.9 5.6 6 PMSFE medium 4.6 5 5.6

[1001] The tested strains can grow on media exclusively made of malt sprouts extracts. One can therefore consider total or partial substitution of conventional sources of nitrogen by malt sprout extracts.

Additional Results

[1002] The result obtained with PMSFE on the growth for three various strains of lactic acid bacteria are good. Each strains tested are able to use the PMSFE as a substitute product to replace the nitrogen source and also may be a part of the sugar source.

[1003] With Lactococcus acidophilus the PMSFE brings something which extends the growth and the results prove that PMSFE extract seems to be well adapted for the growth. Moreover the lactic acid concentrations seem to be highly correlated to the total carbon concentration and in our case with the use of PMSFE extract.

[1004] The using of PMSFE brings a high benefit to the growth of Lactobacillus delbrueckii subsp bulgaricus (every trials with PMSFE are significantly better than the controls) but also for the viability of this strain.

[1005] However, the Lactobacillus strain especially Lactobacillus acidophilus requires compound in yeast extract for his growth. A partial substitution may be considered.

Example 5: Preparation of a Liquid Malt Sprout Fraction Extract from Optimized Diffusion Process

[1006] Malt sprout fraction used to produce this liquid malt sprout fraction extract is characterized in tables 5 and 6 (MSFE2)

[1007] An aqueous extract from MSFE2 is prepared according to the following process. A malt sprout fraction suspension and osmotic water in the weight ratio of 1/10 in order to make diffusion. The said diffusion was carried out at 20 C., during 2 hours. At the end of the 2 hours, a finishing step is carried out as follows: pH adjustment is realized at pH=8, and the aqueous extract is heated at 100 C. for 20 minutes.

[1008] Then a first liquid-solid separation step is realized on aqueous extract, by centrifugation (RC12BP+at 13000 g, 30 min, 20 C.) in order to remove all insoluble particles.

[1009] Characterization of clarified and final aqueous extract is detailed in table 16.

TABLE-US-00019 TABLE 16 Characterization of liquid malt sprout fraction extract (MSFE2L) obtained from optimized diffusion process: MSFE2L (Optimized diffusion process) Dry matter dm (%) 1.5 Total nitrogen (g/kg dm) 50.6 Total FAN (g/kg dm) 15.70 Total DAN (g/kg dm) 13 Total PUPY (g/kg dm) 10.86 Ratio PUPY/DAN 83.5

Example 6: Effect of Malt Sprout Fraction Extract in Syrup Form (Also Named Syrup or Slurry Concentrate and MSFE2) in Lactic Acid Bacteria Culture Media

[1010] The MSFE2 as prepared in example 2 and characterized in table 8 was used in the following test. These cultures were performed on agar plates, static at 37 C. under controlled atmosphere Candle jars. Three types of media were used: [1011] MRS (same references than in examples 4.3.1 and 4.3.2), [1012] MRS media recomposed from commercial sources of nitrogen, [1013] media consisting of syrup in substitution of the total nitrogen sources EFM
5 drops of 0.1 l of different cultures are deposited on the surface of each agar media box. Growth is measured each day for 3 days.

TABLE-US-00020 TABLE 17 Culture of L.acidophilus vs MRS media on agar media - y comparison of colony diameter (in millimeters mm). L acidophilus 18 h 24 h 42 h MRS 7.06 7.07 7.24 MRS recomposed 6.88 7.10 7.3 EFM medium 6.31 6.32 6.48

[1014] The tested strain can grow also on media exclusively made of malt sprouts extracts in powder form (Example 4, Table 17), and in liquid concentrated form (example described here). One can therefore consider total or partial substitution of conventional sources of nitrogen by malt sprout extracts.

Example 7: Interest of the Finishing Step in Combination with Diffusion or Hydrolysis

[1015] Malt Sprout Extracts Preparation (MSFE 2, 3, 4): [1016] a) Mixing of 1 part of malt sprout fraction with 10 parts of water (=slurry) in a tank with stirring device. [1017] b) Diffusion of the solubles into water during 2 h hours at ambient temperature (20 C.) OR diffusion of the solubles into water during 2 h hours at 50 C. OR hydrolysis with Sumizyme LP and Protease Plus (at 0.3% of malt sprout fraction dry matter). [1018] c) After 2 or 5 hours, apply or not he following heat treatment: adjustment of the pH slurry at 8 and heating up to boiling point (100 C.) during 5 min. [1019] d) The slurry is then centrifuged at 13000 g during 10 min. [1020] e) The supernatant is then recovered and constitutes the malt sprout fraction extract.

TABLE-US-00021 Ref MSFE 2 MSFE 3 MSFE 4 Process used to Diffusion at Hydrolysis at Hydrolysis at prepare MSFE 20 C.-2 h 5 h-50 C. 5 h-50 C. Malt sprouts 18 29 29 fractions protein content % N % Malt sprouts 2.97 4.64 4.64 Finishing step pH 8 - 100 C.- No pH 8 - 100 C.- 20 mn 20 mn DM in extract % WV 1.47 4.2 4.65 DAN mg/L extract 177 399 379 PUPY mg/L extract 92.45 355 317 PUPY/DAN 0.52 0.89 0.84 Nt g/L extract 0.7 2.9 3 FAN mg/L extract 141* 702 685 *Estimated OBSERVATIONS/CONCLUSIONS: In those experiments we searched to find extractions conditions giving concomitantly high concentrations of soluble extracts (DM in extract %) and DAN, and high ratios of PUPY/DAN. Nt (total nitrogen in the extracts) and FAN are given as indicators of N and organic N compounds enrichment.

[1021] With selected malt sprout fraction (29% crude protein) hydrolysis alone and hydrolysis with finishing treatment allow to obtain concomitantly high concentrations of soluble extracts (respectively 4.2 and 4.65%, high concentrations of DAN (respectively 399 and 379 mg/L) and PUPY (355 and 317 mg/L) and PUPY/DAN ratio above 80% (respectively 0.89 and 0.84). However, DAN, PUPY and FAN concentrations in MSFE 4 are lower than in MSFE 3 although DM in extract is higher. It is an indication that 20 mn heat treatment is too severe after 5 h hydrolysis at 50 C. and result in losses of organic nitrogen compounds. Heat treatment conditions should be further optimized in relation with hydrolysis conditions to fine tune the best extraction processes.

[1022] These experiments show that it is possible to obtain extensive hydrolysis of nucleic acids and high concentrations of soluble extracts and that further optimization is feasible.

Example 8: Growth of Lactic Acid Bacteria in Model Fermentation Media Demonstrate the Effect of PUPY Supplementation on Bacterial Growth

[1023] 1. Fermentation Media Ingredient Characterization

TABLE-US-00022 Total FAN DAN PUPY Supplier/ nitrogen (N) g/kg g/kg g/kg Ingredient Used as product code g/kg dm dm dm dm Soy Source of Solabia/ 98.9 24.73 1.65 0.37 peptone nitrogen Soy peptone F-A1603

[1024] The pure purines and pyrimidines used in this experiment were supplied by Sigma-Aldrich under the following product references: A8626, C3506, U0750, G11950, H9377. These products have a purity >99%.

[1025] 2. Fermentation Parameters

[1026] Equipment: [1027] Microplate reader TECAN Infinite 200 Pro, connected to computer using Magellan or Icontrol software. [1028] 96 wells microplate

[1029] Measured Parameter:

[1030] Optical density (OD) at 600 nm wavelengthTo measure bacterial concentration.

[1031] Fermentation Kinetics Parameters: [1032] 12 h pre-culture in MRS broth (from DIFCO) [1033] Total volume/well: 200 L [1034] Percent inoculum addition: 10% [1035] Number of replicates=3 [1036] Stirring: 3 mm of orbital amplitude during 15 minutes [1037] Temperature=30 or 37 C. depending on the strain [1038] Follow-up over 24 hours with measurement every 30 minutes Strains [1039] Lactobacillus helveticus LAFTI L10 (cultivated at 37 C.) [1040] Lactobacillus plantarum ATCC 14917 (cultivated at 30 C.) [1041] Pediococcus pentosaceus ATCC 33316 (cultivated at 30 C.)

[1042] 3. Fermentation Media Formulation

[1043] Three Basic Solutions were Prepared in Order to Produce 4 Media:

TABLE-US-00023 PUPY concentration FAN concentration Media (mg/L) (mg/L) 1 0 390 2 0 780 3 150 390 4 150 780 [1044] A base medium composed of glucose and mineral salts: 500 mL at 120.6 g/L (M)

TABLE-US-00024 Concentration Mass for 500 mL Ingredients (g/L) solution (g) Glucose 80 40 Tween 80 4 2 Dipotassium phosphate K2HPO4 8 4 Sodium acetate C2H3NaO2 20 10 Ammonium citrate C6H5 + 4yFexNyO7 8 4 Magnesium sulfate MgSO4 0.4 0.2 Manganese sulfate MnSO4 0.2 0.1 [1045] A nitrogen solution composed of soy peptone: 300 mL at 60 g/L (N)

TABLE-US-00025 Concentration Mass for Ingredient (g/L) 300 mL (g) Soy peptone 60 18 [1046] A PUPY solution composed of pure purines and pyrimidines bases: 800 mL at 3 g/L (PUPY)

TABLE-US-00026 Concentration Mass for 800 PUPY % (g/L) mL (g) C 3 0.09 0.0720 H 55 1.65 1.3200 U 25 0.75 0.6000 G 8 0.24 0.1920 A 9 0.27 0.2160 Total 100 3.00

[1047] Final fermentation medium formulation:

TABLE-US-00027 Media Volume (M) Volume (PUPY) Volume (N) Volume (water) Total volume 1 25 mL 0 mL 25 mL 50 mL 100 mL 2 25 mL 0 mL 50 mL 25 mL 100 mL 4 25 mL 5 mL 25 mL 45 mL 100 mL 3 25 mL 5 mL 50 mL 20 mL 100 mL

4. Results

[1048]

TABLE-US-00028 Maximum Absorbance (OD at 600 nm) PUPY 0 mg/L PUPY 150 mg/L Strains FAN 390 FAN 780 FAN 390 FAN 780 mg/L mg/L mg/L mg/L L. plantarum ATCC 14917 1.367 1.506 1.608 1.578 P. pentosaceus ATCC 33316 1.425 1.426 1.563 1.531 L. helveticus LAFTI L10 1.357 1.550 1.547 1.436

[1049] In the non-supplemented media (PUPY=0) the bacteria can find available FAN to support their growth and the maximum concentration of biomass is obtained at the highest concentration of FAN.

[1050] In the supplemented media (PUPY=150), the bacteria can find the available FAN to support their growth, but the maximum concentration of biomass (Amax) is obtained at the lowest concentration of FAN (390 mg/L). The maximum concentrations of biomass are obtained at FAN=390 mg/L and DAN=150 mg/L. However, for L helveticus, Amax is practically equal to Amax obtained at FAN 780-DAN 0, and the highest Amax are obtained for L plantarum and P pentosaceus at FAN390-DAN 150. This unexpected result is particularly interesting because it shows complementarity and or synergy between FAN and DAN.

Example 9: The Use of Malt Sprout Fraction Extract as Substitute to Conventional Nitrogen Sources in Media Formulation for Lactic Acid Bacteria

[1051] The standard media to grow lactic acid bacteria (of genus Lactobacilli) is MRS broth. This media is constituted of various nitrogen sources: proteose-pepton, Meat extract, and yeast extract.

[1052] In these experiments, MSFE is used as substitute of all nitrogen sources (w/w) and of each of the nitrogen sources.

[1053] 1. Characterization of Nitrogen Sources

TABLE-US-00029 FAN FAN DAN PUPY g/kg (g/L) g/kg DAN g/kg PUPY Quantity dm in in dm in (g/L) in dm in (g/L) in N (g) in 55 N media N media N media sources g dm source 55 g/L source 55 g/L source 55 g/L DAN/FAN PUPY/FAN 1 10 12.24 0.1224 4.62 0.0462 2.05 0.0205 0.377 0.1675 Proteose- pepton 2 Beef 10 19.96 0.1996 2.44 0.0244 0.911 0.00911 0.1222 0.0456 meat extract 3 Yeast 5 36.1 0.1805 7.93 0.03965 1.6 0.008 0.2197 0.0443 extract MSFE 10.58 5.965 5.387 0.564 0.509 022204

[1054] 2. Media Formulations

TABLE-US-00030 FAN FAN DAN PUPY g/kg (g/L) g/kg DAN g/kg PUPY Quantity dm in in dm in (g/L) in dm in (g/L) in N (g) in 55 N media N media N media sources g dm source 55 g/L source 55 g/L source 55 g/L DAN/FAN PUPY/FAN Medium 1 10 10.58 0.1058 5.965 0.05965 5.387 0.05387 MSE substitutes Proteose pepton 2 + 3 15 0.3801 0.06405 0.01711 Medium 1 55 0.4859 0.1237 0.07098 0.255 0.146 Medium 2 10 10.58 0.1058 5.965 0.05965 5.387 0.05387 MSE substitutes meat extract 1 + 3 15 0.3029 0.08585 0.0285 Medium 2 55 0.4087 0.1455 0.08237 0.356 0.202 Medium 3 5 10.58 0.0529 5.965 0.029825 5.387 0.026935 MSE substitutes yeast extract 1 + 2 20 0.322 0.0706 0.02961 Medium 3 55 0.3749 0.100425 0.056545 0.296 0.151 Control 25 0.5025 0.11025 0.03761 0.219 0.075 medium No substitution 1 + 2 + 3

[1055] 3. Impact of the Substitution of Conventional Nitrogen Sources on the Maximum Concentration (Amax) of Different Lactic Acid Bacteria

TABLE-US-00031 A max in A max in CONTROL SUBSTITUTED SUBSTITUTED Improvement Strains MEDIUM MEDIUM MEDIUM factor Lactobacillus 0.452 Medium 2 0.922 2.04 acidophilus ATCC 4356 Lactobacillus 0.449 Medium 3 0.501 1.12 delbrueckii lactis ATCC 1215 Lactobacillus 1.482 Medium 1 1.594 1.08 helveticus LAFTI L10

[1056] Depending on the N compounds requirements of the strains, MSE can substitute N sources like proteose-pepton, meat extract or yeast extract with improved final biomass concentration. It can be observed that all the substituted media have a lower concentration of FAN than the control medium but higher PUPY concentration and PUPY/FAN ratio. These very surprising results show that MSE offer the unique possibility to reduce FAN requirements when proper levels of DAN and PUPY are present in the media. It is particularly interesting to optimize performances of fermentations.

Example 10: The Use of Malt Sprout Fraction Extract as Flavor Enhancer in Chicken Broth

[1057] 1. Characterization of Malt Sprout Extract Used in the Experiment.

TABLE-US-00032 Dry Total FAN DAN PUPY Ratio matter nitrogen g/kg g/kg g/kg PUPY/DAN Used as Form % (N) g/kg dm dm dm dm % MSFE Flavor Powder 95 66.4 9.71 6.67 6.41 96.1 ORG2205 enhancer

[1058] 2. Basic Matrix: Homemade Chicken Broth

[1059] For the production of about 1 liter of chicken broth

[1060] Ingredients: [1061] 1 chicken carcass (cut into pieces)for an average chicken about 300 g. [1062] 1 carrotapprox. 100-120 g [1063] 1 celery stalkapprox. 40 g [1064] 1 white leekapprox. 120 g [1065] 1 yellow onionapprox. 100 g [1066] 1 branch of thyme2 g [1067] 1 branch of laurel4-5 medium leaves [1068] 1 pinch of salt3 g [1069] 1.2 liters of water

[1070] Recipe:

[1071] 1. Peel and cut into coarse pieces carrot, celery stalk and leek.

[1072] 2. Cut the onion into 4.

[1073] 3. In a casserole, fry the pieces of chicken carcass until lightly browned.

[1074] 4. Add the carrot, celery and leek, as well as the onion.

[1075] 5. Cover with water

[1076] 6. Add the pinch of salt, thyme and bay leaf.

[1077] 7. Simmer without cover for about 1 hour without boiling.

[1078] 8. When cooking is complete, let everything cool in the pan.

[1079] 9. Once the mixture has cooled, filter with a strainer to recover the broth.

[1080] 10. Mix the different productions to obtain a single batch.

[1081] 11. Store covered broth in refrigerator ( day).

[1082] 12. Before freezing the juice, remove the layer of fat that is on the surface (degreasing).

[1083] 4 concentrations of the malt sprout fraction extract have been tested: 0% 0.5% 0.8% 1% (g/100 g of chicken broth).

[1084] 3. Sensory Profile Methodology [1085] a) Methodology: Sensory profile [1086] NF EN ISO 13299 May 2010 (Sensory analysis-Methodology-General guidelines for the establishment of a sensory profile) [1087] NF EN ISO 8586 February 2014 (Sensory analysis-General guidelines for the selection, training and control of qualified subjects and expert sensory subjects) [1088] b) Protocol [1089] 15 people were recruited from the external panels trained by SensoStat. [1090] 5 training sessions of approximately 1 hour were conducted over 1 month. The panel was therefore qualified to analyze the type of product studied. [1091] The subjects were trained on a list of descriptors determined in agreement with Malteurop and with their feelings during the sessions, using classifications and identification of flavors and aromas, scale tests, presentation of references, group discussions on different descriptors. The products used in this phase were purchased commercially or consisted of homemade chicken broth with different concentrations of the test compound. [1092] The study took place in the premises of the Center for Taste and Food Sciences (Dijon), in individual tasting booths, under standardized light conditions and temperatures. [1093] The cabins are computerized to automate data acquisition (FIZZ). [1094] The cabins were lit up in white light. [1095] The training sessions took place in individual cabins or in a meeting room when a discussion was needed. [1096] c) Measurement sessions [1097] The measurement sessions took place on Dec. 1, 2017. The 4 concentrations were evaluated on the list of 6 descriptors: [1098] Vegetables [1099] Chicken [1100] Umami [1101] Salt [1102] Aromatic herbs [1103] Fat [1104] The products were evaluated on continuous scales from 0 to 10 [1105] The evaluation of the 4 recipes was made with the monadic order of presentation following a Latin square, with a repetition (each recipe was evaluated twice by each subject). [1106] The products were served blind, in black cups (to avoid visual differences) coded (about 20 ml), at about 45 C. [1107] A 2-minute break was observed between each sample. Rinsing was done with water and unsalted crackers to optimize mouth cleaning.

[1108] 4. Results [1109] a) Analyse of variance: product effect

TABLE-US-00033 P value Products: 0% 0.5% 0.8% 1% Product effect Vegetable 7.53 7.603 7.547 7.107 0.579 Chicken 7.363 a 7.337 ab 6.643 bc 6.467 c 0.082 * Umami 6.477 b 6.587 b 7.267 a 7.353 a 0.064 * Salt 6.333 b 6.21 b 7.1 a 6.733 ab 0.042 ** Aromatic herbs 5.637 5.69 6.23 6.313 0.319 Fat 1.653 b 1.527 b 2.217 a 1.907 ab 0.080 *

[1110] Anova model: subject+product

[1111] Fisher test (LSD)/Analysis of differences between products with a 90% confidence interval

[1112] Products linked by the same letter are not statistically different [1113] b) Observations: [1114] 0.8% and 1% samples: [1115] Higher intensity on Umami, Salty and Fat (statistically significant differences) [1116] Lower Intensity on Chicken (statistically significant difference) [1117] No effect of the test compound on the descriptors Vegetables and Aromatic herbs. [1118] c) Conclusions

[1119] The 0.8% and 1% samples have a higher intensity than the 0.5% and 0% products on the Umami, Salty and Fat descriptors. They have a lower intensity on the Chicken descriptor.

[1120] No effect of the test compound was demonstrated on the Vegetable and Aromatic herb descriptors.

[1121] We can Therefore Conclude that at 0.8% and 1%, the Studied Compound has an Enhancing Effect on Umami and Salty, and on a Fat Taste.

Example 11: Comparative Composition of Standard Malt Sprout Extract and Malt Sprout Fraction Extracts Prepared According to the Invention

[1122] Preparation of Malt Sprout Extract (MSE) and Malt Sprout Fraction Extract (MSFE) and Characterization of the Compositions of the Resulting Extracts.

[1123] Different methods or processes including raw material selection to prepare malt sprout extracts are described in some published documents. Very limited information is available on the characteristics of the resulting extracts. The compositions are poorly defined. The in-depth research works that the inventors have carried out on MSFE demonstrate the importance of nucleic acid hydrolysis to obtain defined DAN and PUPY concentrations in MSFE and a defined ratio of PUPY/DAN.

[1124] Nothing is published in the prior art on the concentrations of DAN and PUPY in malt sprouts extracts obtained with the different existing methods or processes. These methods or processes including raw material selection differ from one to another but the absence of knowledge on DAN and PUPY concentrations, or other relevant components, in the respective extracts does not allow to differentiate the composition of these extracts.

[1125] 1. Preparation of Standard Malt Sprout Extract (MSE) and Malt Sprout Fractions Extracts (MSFE)

[1126] For this experiment, a malt sprout extract (MSE) was prepared according to a conventional method using common malt sprouts (18-23% crude proteins) from a usual malting process. The resulting extract (MSE c) was used as a control to define its composition.

[1127] Two malt sprout fraction extracts (MSFE) were prepared from a selected malt sprout fraction (MSF4) obtained from the same common malt sprout. The selected malt sprout fraction contained more than 80% rootlets and acrospires (and between 28 and 34% crude proteins).

[1128] One malt sprout fraction extract (MSFE4-A) was obtained using a diffusion method followed by a finishing step. A second malt sprout fraction extract (MSFE4-B) was obtained using a hydrolysis method in the presence of exogenous enzymes, followed by a finishing step.

[1129] Both MSFE4-A and MSFE4-B, obtained according to the claimed methods of preparation, were analyzed to define the compositions and compare with MSE c.

[1130] The remaining solid residues resulting from the 3 methods of preparation were also recovered and analyzed to determine the compositions and to establish the respective overall mass balance and Nitrogen components mass balances.

[1131] 2. Comparative Analytical Characterizations of MSE and MSFE

[1132] The analytical work included:

[1133] Analysis of common malt sprout and selected malt sprout fraction including: dry matter, extract, Nt, crude protein, soluble protein, FAN, diastasic power, nucleic acids, DAN, PUPY, pentoses, crude fibers, soluble and insoluble fibers, soluble -glucans, starch, total sugars, fats, ashes.

[1134] Analysis of extracts MSE c, MSFE4-A and MSFE4-B including: soluble dry matter, Nt, crude protein, soluble protein, FAN, amino acids, diastasic power, oligonucleotides, DAN, PUPY, pentoses (xylose, arabinose, ribose, deoxyribose), glucose, crude fibers, soluble and insoluble fibers, soluble -glucans, starch, total sugars, fats, ashes.

[1135] Analysis of remaining solid residues including: wet mass after rinsing, dry matter, Nt, crude protein, soluble protein, FAN, diastasic power, nucleic acids, DAN, PUPY, pentoses, crude fibers, soluble and insoluble fibers, soluble -glucans, starch, total sugars, fats, ashes.

[1136] Compositions of the 3 extracts are compared to establish critical difference in selected components and the advantages of the claimed extracts and methods.