Sugar-dipeptide conjugates as flavor molecules

Abstract

The present invention relates to compounds and compositions for use in enhancing flavor and umami taste of food products. Particularly, the present invention relates to compounds of the general formula I) and compositions comprising them. ##STR00001##

Claims

1. A composition comprising the compound of the general formula I, ##STR00003## wherein R1 is selected from the group consisting of a hydrogen, a C.sub.1, a C.sub.2, a C.sub.3, and a C.sub.4 alkyl group, or a salt of the compound, the composition is selected from the group consisting of a culinary seasoning, a sauce or soup concentrate, and dry or wet pet-food product, and a final concentration of the compound in the composition is 1 mg/g to 3 mg/g.

2. The composition according to claim 1, wherein the composition is an extract from plant and/or meat material.

3. The composition according to claim 1, wherein the composition is the result of a flavor reaction.

4. A method for enhancing the flavor and/or umami taste of a culinary food product, the method comprising adding a compound of the general formula I, ##STR00004## wherein R1 is selected from the group consisting of a hydrogen, a C.sub.1, a C.sub.2, a C.sub.3, and a C.sub.4 alkyl group, or a salt of the compound to the culinary food product, and a final concentration of the compound in the culinary food product is 1 mg/g 3 mg/g.

5. The composition of claim 1, wherein the final concentration of the compound in the composition is 1 mg/g to 2.5 mg/g.

6. The composition of claim 1, wherein the final concentration of the compound in the composition is 1 mg/g to 2.0 mg/g.

7. The method of claim 4, wherein the final concentration of the compound in the culinary food product is 1 mg/g to 2.5 mg/g.

8. The method of claim 4, wherein the final concentration of the compound in the culinary food product is 1 mg/g to 2.0 mg/g.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: HPLC-UV chromatogram of a thermally treated carnosine with glucose.

(2) FIG. 2: HPLC-UV chromatogram of a thermally treated anserine nitrate salt with glucose.

DETAILED DESCRIPTION OF THE INVENTION

(3) The present invention pertains to a compound of the general formula I), wherein R1 is a hydrogen, a C.sub.1, a C.sub.2, a C.sub.3, or a C.sub.4 alkyl group; or a salt of said compound. In one embodiment, the R1 group of the compound of the present invention is a hydrogen or a methyl group. The chemical names of those corresponding two compounds are: 1-deoxy-D-fructosyl-N--alanyl-L-histidine and 1-deoxy-D-fructosyl-N--alanyl-N-methyl-L-histidine, respectively.

(4) A second aspect of the invention relates to a composition comprising said compound of the general formula I) in an amount of at least 1 mg/g, at least 1.7 mg/g, at least 2 mg/g, at least 2.5 mg/g, at least 3 mg/g, at least 3.5 mg/g, or at least 5 mg/g of the total composition.

(5) In one embodiment, the composition of the present invention is an extract from plant and/or meat material. For example, the composition is an extract from beef meat, chicken meat, pork meat or a combination thereof.

(6) In another embodiment, the composition of the present invention is the result of a flavor reaction. The term flavor reaction refers herein to a chemical reaction occurring between at least one reducing sugar and at least one amino acid or protein. Typically, this chemical reaction occurs during a heating process and is typically also referred to as Maillard reaction. In one example, the flavor reaction is a Maillard reaction.

(7) In a preferred embodiment, the composition of the present invention is food grade. Under food grade the inventors mean that the composition is suitable for human consumption, for example directly, in concentrated form, and/or when used diluted in a food product.

(8) For example, the composition of the present invention is selected from the group consisting of a culinary seasoning product, a cooking aid, a sauce or soup concentrate, a dry or wet pet-food product.

(9) Further aspects of the present invention relate to a use of said compound for enhancing the flavor and/or the umami taste of a food product. Such a food product may be a ready-to-eat food product. It may also be a flavor concentrate used for seasoning a still further other food product. Advantageously, the compound of the present invention may be used for being added to a seasoning, a cooking aid or a food concentrate product. Thereby the strength of providing an umami taste to a still further food product is improved in such a seasoning, cooking aid or food concentrate product.

(10) Further aspects of the present invention also relate to a use of a composition comprising said compound in an amount of at least 1 mg/g, at least 1.7 mg/g, at least 2 mg/g, at least 2.5 mg/g, at least 3 mg/g, at least 3.5 mg/g, or at least 5 mg/g of the total composition, for enhancing the flavor and/or the umami taste of a food product. Advantageously, such a food product may be a ready-to-eat food product. The use of the present invention has the advantage that it allows to use natural extracts which for example have been enriched in said compounds for flavoring and improving the natural umami taste of those food products.

(11) A still further aspect of the present invention is a method for enhancing the flavor and/or umami taste of a culinary food product, comprising the step of adding said compound or the composition comprising said compound to a food product. The food product can be a ready-to-eat food product or a flavor concentrate.

(12) As an example of the present invention, the final concentration of said compound in the food product is at least 1 mg/g, at least 1.7 mg/g, at least 2 mg/g, at least 2.5 mg/g, at least 3 mg/g, at least 3.5 mg/g, or at least 5 mg/g of the composition. This advantageously, allows generating for example food seasoning products and flavor concentrate products which convey a strong umami taste to a further food product upon application.

(13) Those skilled in the art will understand that they can freely combine all features of the present invention disclosed herein. In particular, features described for the products of the present invention may be combined with the uses and method of the present invention, and vice versa. Further, features described for different embodiments of the present invention may be combined.

(14) Further advantages and features of the present invention are apparent from the figures and examples.

EXAMPLE 1

Synthesis of 1-deoxy-D-fructosyl-N--alanyl-L-histidine from Glucose and Carnosine (-alanyl-L-histidine)

(15) Chemicals: Sodium bisulphite and glycerol were purchased from Sigma, glucose from SDfine Chemicals, carnosine from ChemImprex, methanol and acetic acid from Merck. All commercially available reagents were used as received, from their respective suppliers.

(16) Analytical thin layer chromatography (TLC) was carried out on RP-18 F254s (Merck) plates. The TLC plates were visualized by shortwave UV light, Ninhydrin stain.

(17) .sup.1H NMR (360.13 MHz) and .sup.13C NMR (90.56 MHz) spectra were recorder on a Bruker DPX-360 spectrometer equipped with a broadband multinuclear z-gradient probehead. The chemical shifts (in ppm) were expressed with respect to an internal reference (TMS or TSP). Multiplicities are reported as follows: s=singlet, d=doublet, t=triplet, q=quadruplet, m=multiplet, bs=broad singlet.

(18) D-Glucose (23 g, 127.37 mmol, 2.8 eq.) and sodium bisulfite (1.6 g, 12.389 mmol, 0.28 eq.) were suspended in methanol (38 mL) and glycerol (19 mL). After stirring for 30 min at 100 C., carnosine (10 g, 44.22 mmol, 1.0 eq.) and acetic acid (5.1 mL) were added and the resulting mixture was heated for 3.5 hours at 100 C. Reaction mass was then cooled down and diluted with 38 mL water. The reaction mixture was purified using a column packed in Amberlite IRN-77 ion exchange resin (100 g). NH.sub.3 0-0.4% was used as gradient in water for elution. Finally, 6.8 g 1-deoxy-D-fructosyl-N--alanyl-L-histidine were isolated (39.62%); Rf (n-Butanol:Acetic Acid:Water, 3:2:2): 0.21; MS (M.sup.+): m/z 388.16; .sup.1H NMR (Deuterium Oxide) 2.77 [m, 2H], 3.13 [dd, J=15.4, 8.2 Hz, 1H], 3.21-3.27 [m, 1H], 3.28-3.32 [m, 2H], 3.33-3.44 [m, 2H], 3.63-3.75 [m, 1H], 3.76-3.85 [m, 2H], 3.87-3.91 [m, 1H], 3.99-4.03 [m, 2H], 4.53 [dd, J=8.2, 5.2 Hz, 1H], 7.28 [d, J=1.0 Hz, 1H], 8.61 [d, J=1.4 Hz, 1H]; .sup.13C NMR (Deuterium Oxide) 26.98, 30.26, 44.28, 53.01, 53.92, 63.91, 68.80, 69.20, 69.76, 95.21, 116.65, 129.49, 133.15, 171.60, 176.13.

EXAMPLE 2

Preparation of 1-deoxy-D-fructosyl-N--alanyl-L-histidine from Glucose and Carnosine (-alanyl-L-histidine)

(19) A mixture of carnosine (226 mg; 1 mmol; 1 eq.) and glucose (360 mg; 2 mmol; 1 eq.) in 20 mL Na.sub.2HPO.sub.4 buffer (0.5 mol/L, pH 7.0) was heated in a closed vessel at 80 C. for 3 h. The solvent was then evaporated under reduced pressure and the resulting precipitate was freeze-dried. Aliquots of the freeze-dried powder were dissolved in water upon ultrasonification for 10 min and filtrated (0.45 m). The solutions were then fractionated by a semi-preparative hydrophilic interaction liquid chromatography (HILIC-HPLC) using a 30021.5 mm i.d., 10 m, TSKgel Amide-80 column (Tosoh Bioscience, Stuttgart, Germany) equipped with a 7521.5 i.d., 10 m, guard column (Tosoh Bioscience, Stuttgart, Germany). Monitoring the effluent with an ELSD detector (Evaporative Light Scattering Detector) and adjusting the flow rate to 8 mL/min, a gradient consisting of aqueous formic acid (1% in water, solvent A) and acetonitrile (solvent B) was used. Starting with a mixture of 75% B and 25% A for 10 min, the gradient was reduced successively to 0% B and 80% A within another 10 min. After holding these conditions for 5 min, the gradient was increased to 75% B and 25% A within 8 min. The purification led to 6 fractions as shown in the FIG. 1.

(20) The molecule corresponding to fraction F5 was identified as carnosine while the molecule F6 was identified as 1-deoxy-D-fructosyl-N--alanyl-L-histidine (based on LC-MS and NMR data).

EXAMPLE 3

Preparation of 1-deoxy-D-fructosyl-N--alanyl-L-histidine from Glucose and Carnosine (-alanyl-L-histidine)

(21) A mixture of carnosine (905 mg; 4 mmol) and potassium hydroxide (224 mg; 4 mmol) in 100 mL methanol was refluxed for 2 h. After cooling down to room temperature, the precipitate was removed by filtration and the supernatant was concentrated under reduced pressure leading to the carnosine potassium salt. Then, a mixture of the carnosine potassium salt (2 mmol) and glucose (360 mg; 2 mmol) in methanol (50 mL, pH 5.0 with formic acid) was heated at 80 C. in a closed vessel for 2 h. After evaporating the solvent under reduced pressure, the precipitate was dissolved in water and freeze-dried. The reaction product was purified using same conditions as reported in the Example 2.

EXAMPLE 4

Preparation of 1-deoxy-D-fructosyl-N--alanyl-N-methyl-L-histidine from Glucose and Anserine (-alanyl-N-methyl-L-histidine)

(22) D-Glucose (127.37 mmol, 2.8 eq) and sodium bisulfite (12.389 mmol, 0.28 eq) were suspended in methanol (38 mL) and glycerol (19 mL). After stirring for 30 min at 100 C., anserine nitrate salt (44.22 mmol, 1.0 eq, Bachem) and acetic acid (5.1 mL) were added and the resulting mixture was heated for 3.5 hours at 100 C. Reaction mass was then cooled down and diluted with water (38 mL). The mixture was purified by preparative liquid chromatography using Phenomenex Luna 5HILIC 2504.60 mm column with 5 mM NH.sub.4Ac buffer in water (solvent A) and acetonitrile (90%, solvent B), adjusted to a pH 5.8. The resulting chromatogram and the gradient are presented in FIG. 2. The first peak was identified as the nitrate salt while peak 2 is the desired 1-deoxy-D-fructosyl-N--alanyl-N-methyl-L-histidine and peak 3 corresponds to the unreacted anserine. Finally, 2.6 g 1-deoxy-D-fructosyl-N--alanyl-N-methyl-L-histidine were isolated (15%).

(23) LC-MS (ESI.sup.): m/z 401.13 (100, [M-H]).sup.; .sup.1H NMR (400 MHz, 300 K, Deuterium Oxide) 2.70-2.86 [m, 2H], 3.09 [dd, J=15.8, 8.3 Hz, 1H], 3.26 [dd, J=15.8, 5.0 Hz, 1H], 3.34-3.40 [m, 2H], 3.36 [m, 2H], 3.71-3.77 [m, 1H], 3.78-3.83 [m, 1H], 3.85 [s, 3H], 3.88-3.92 [m, 1H], 3.99-4.05 [m, 2H], 4.51 [dd, J=8.5, 5.5 Hz, 1H], 7.24 [s, 1H], 8.54 [s, 1H]; .sup.13C NMR (100 MHz, 300 K, Deuterium Oxide) 28.77, 33.28, 35.87, 47.24, 55.96, 56.18, 66.85, 71.74, 72.14, 72.68, 98.18, 120.99, 133.96, 138.25, 174.55, 179.19.

EXAMPLE 5

Sensory Evaluation of Carnosine (-alanyl-L-histidine) and Anserine (-alanyl-N-methyl-L-histidine) in Model Broth

(24) The sensory tests were performed in a sensory panel room at 20-25 C. To avoid a retro-nasal aroma or taste impression, nose clips were used. The sensory panel consisted of 8 to 14 trained persons. The panel was trained to evaluate the taste of aqueous solutions (1 mL each) of the following standard taste compounds by using a triangle test: saccharose (50 mmol/L) and L-alanine (15 mmol/L), respectively, for sweet taste; lactic acid (20 mmol/L) for sour taste; NaCl (12 mmol/L) for salty taste; caffeine (1 mmol/L) and quinine hydrochloride (0.05 mmol/L), respectively, for bitter taste; sodium glutamate (8 mmol/L, pH 5.7) for umami taste; and tannin (0.05%) for astringency. The white meaty oral sensations was assessed in a model broth solution prepared from monosodium glutamate monohydrate (1.9 g/L), yeast extract (2.1 g/L), maltodextrin (6.375 g/L) and sodium chloride (2.9 g/L) in bottled water (pH 5.9).

(25) The taste threshold concentration of -alanyl-L-histidine was determined in the model broth using a three-alternative test with two blanks and one sample in ascending concentrations of -alanyl-L-histidine. The taste threshold concentration was found to be 22700 mol/L (5.3 mg/g) for the thick-sour sensation and white-meaty oral impression.

(26) The taste threshold concentration of -alanyl-N-methyl-L-histidine can be determined in the same way as described above for the -alanyl-L-histidine.

EXAMPLE 6

Sensory Evaluation of 1-deoxy-D-fructosyl-N--alanyl-L-histidine and 1-deoxy-D-fructosyl-N--alanyl-N-methyl-L-histidine in Model Broth

(27) The taste threshold concentration of 1-deoxy-D-fructosyl-N--alanyl-L-histidine was determined in model broth as described in Example 5 and was found to be 4400 mol/L (1.7 mg/g) for the thick-sour sensation and white-meaty oral impression. This taste threshold value is considerably lower than the 22700 mol/L (5.3 mg/g) threshold level determined for the corresponding -alanyl-L-histidine under the same experimental model system (see Example 5). In fact, it corresponds to a lowering of the taste threshold concentration by a factor of about 5.

(28) This result means that about a 5-time smaller amount of molecules of 1-deoxy-D-fructosyl-N--alanyl-L-histidine is required to impart a same corresponding taste impact of flavour and umami taste enhancement in a food product than with the corresponding -alanyl-L-histidine under the same conditions.

(29) A same result and similar quantitative reduction can be observed when testing and comparing the taste threshold values of 1-deoxy-D-fructosyl-N--alanyl-N-methyl-L-histidine in comparison with the corresponding anserine (-alanyl-N-methyl-L-histidine).

EXAMPLE 7

Identification and Quantification of 1-deoxy-D-fructosyl-N--alanyl-L-histidine in Meat-bouillons

(30) Preparation of the Pot-au-feu bouillon: The ingredients (from local market) and their amounts are summarized in Table 1. Meat pieces were suspended in 5 L cold water. 22.5 g NaCl were added and the mixture was boiled. After 2 hours, the vegetable-cuts were added to the bouillon and the preparation was boiled for an additional hour. The mixture was filtered to remove solid parts.

(31) TABLE-US-00001 TABLE 1 Ingredients of the pot-au-feu Vegetables Meat Leek 140 g Flat shoulder 624 g Onions 45 g Round shoulder 62 g Celery 108 g Bone marrow 180 g Navets 88 g Knuckle of veal 190 g Clove 0.35 g Oxtail 303 g Carrots 136 g Flat rips 251 g

(32) Preparation of the meat bouillon: Meat pieces were suspended in 5 L cold water (Table 1). 22.5 g NaCl were added and the mixture was boiled for 3 hours. The mixture was filtered to remove solid parts.

(33) 50 mL bouillons were spiked with a defined amount of .sup.13C.sub.6-labeled standards, applied on a Strata C18-E cartridge and eluted with water to reach an effective dilution of 1:10.

(34) Quantification was done by stable isotopic dilution analysis using a HPLC-MS equipped with TSKgel-Amide 80 column (3 m, 2 mm150 mm from Tosoh Bioscience, Stuttgart, Germany) and the guard column TSKgel-Amide 80 (3 m, 2 mm10 mm from Tosoh Bioscience, Stuttgart, Germany). The eluent A was a mixture of acetonitrile with 1.0% formic acid and the eluent B was water with 1.0% formic acid. The injection volume was 3 L. The flow rate was 0.2 mL/min. The solvent gradient started at 95% A from 0 to 5 min then 95-5% A from 5 to 15 min, 5% A for 10 min, 5-95% from 27 to 30 min. Table 2 summarizes MS conditions.

(35) TABLE-US-00002 TABLE 2 Mass transitions Q1 .fwdarw. Q3 Substance MW [Da] [m/z] DP.sup.a CE.sup.b CXP.sup.c 1-Deoxy-D-fructosyl-N- 388 389 .fwdarw. 305 71 25 4 -alanyl-L-histidine .sup.aDeclustering Potential; .sup.bCollision Energy; .sup.cCell Exit Potential

(36) 1-Deoxy-D-fructosyl-N--alanyl-L-histidine was found at 10 and 7 mol/L in meat and pot-au-feu bouillons. This corresponds to a concentration of about 2.7 and 3.9 g/g broth, respectively.

EXAMPLE 8

Seasoning Compositions

(37) Chicken soups were prepared by dissolving 6 g chicken base powder (detailed recipe shown in Table 3) and 1 g monosodium glutamate in 500 mL hot water. 1-Deoxy-D-fructosyl-N--alanyl-L-histidine or alternatively 1-deoxy-D-fructosyl-N--alanyl-N-methyl-L-histidine was added at 2 g/L.

(38) TABLE-US-00003 TABLE 3 Composition of chicken base powder Ingredient Quantity (%) Chicken Meat powder 30 Starch 1.52 Flavors 2.58 Celery powder 0.50 Garlic powder 0.90 Chicken fat 8.00 Maltodextrine 56.50 Total 100

(39) The sensory evaluation was carried out by 12 panelists, previously screened for their sensory abilities. The panelists were asked to taste a set of 2 chicken soups, one containing no 1-deoxy-D-fructosyl-N--alanyl-L-histidine and one containing 1-deoxy-D-fructosyl-N--alanyl-L-histidine (2 g/L). If sensory differences were observed, the panelists were asked to describe them.

(40) The sensory panel concluded that chicken soups with and without the 1-deoxy-D-fructosyl-N--alanyl-L-histidine were perceived as significantly different and the addition of 1-deoxy-D-fructosyl-N--alanyl-L-histidine significantly increased the boiled chicken and meaty flavours.

(41) The same sensory evaluation was carried out with the chicken soup samples with and without containing 1-deoxy-D-fructosyl-N--alanyl-N-methyl-L-histidine at 2 g/L broth. The sensory panel concluded that chicken soups with and without the 1-deoxy-D-fructosyl-N--alanyl-N-methyl-L-histidine were perceived as significantly different and the addition of 1-deoxy-D-fructosyl-N--alanyl-N-methyl-L-histidine significantly increased the boiled chicken and meaty flavours.

EXAMPLE 9

Seasoning Compositions

(42) Tomato soups were prepared by dissolving in 6 g tomato base powder (detailed recipe shown in the Table 4) in 500 mL hot water. 1-Deoxy-D-fructosyl-N--alanyl-L-histidine or alternatively 1-deoxy-D-fructosyl-N--alanyl-N-methyl-L-histidine was added at 2 g/L to the soups.

(43) TABLE-US-00004 TABLE 4 Composition of tomato soup powder Ingredient Quantity (g) Yeast extract 0.036 White Sugar 0.348 Flavors 0.629 Tomato powder 0.03 Wheat flour 0.562 Corn starch 0.247 Guar gum 0.012 Spices powder 0.071 Maltodextrine 0.038 Sunflower oil 0.022 Total 2

(44) The sensory panel concluded that tomato soups with and without the 1-deoxy-D-fructosyl-N--alanyl-L-histidine or 1-deoxy-D-fructosyl-N--alanyl-N-methyl-L-histidine were perceived as significantly different; and the addition of 1-deoxy-D-fructosyl-N--alanyl-L-histidine and alternatively 1-deoxy-D-fructosyl-N--alanyl-N-methyl-L-histidine increased significantly the savory, spicy notes of those soups.