Lettuce extract

Abstract

To provide an extract for imparting the desired 2-acetyl-1-pyrroline aroma to food, a composition containing such an extract, and a method for producing the extract.SOLUTION: The present invention relates to an extract from lettuce (Lactuca sativa), in particular of stalk lettuce (Lactuca sativa var. angustana), comprising 2-acetyl-1-pyrroline and, optionally, precursors of 2-acetyl-1-pyrroline which has been heat-treated, as well as flavoring compositions comprising such an extract and a method for the preparation of such an extract. The invention further concerns the use of such an extract as a taste or flavor ingredient in a consumer product and a method of enhancing, improving, modifying the taste or flavor of a consumer product by making use of such an extract.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an extract from lettuce (Lactuca sativa), particularly stem lettuce (Lactuca sativa var. angustana), containing 2-acetyl-1-pyrroline and optionally a precursor of 2-acetyl-1-pyrroline, a flavoring composition containing such an extract, and a method for producing such an extract. The present invention further relates to the use of such an extract as a taste or flavor ingredient in consumer products, and to a method for enhancing, improving, or modifying the taste or flavor of consumer products by using such an extract.

[0002] Background technology Rice is the staple food and the main cereal crop for two-thirds of the world's population. China and India are the main rice-producing countries. Aroma is one of the most important characteristics of rice, especially when consumer acceptance is considered as a criterion. Today, consumers are becoming more conscious of the quality of the rice they consume. Consumers often prefer fragrant rice due to its characteristics and pleasant smell. Therefore, the demand for fragrant rice is increasing in both domestic and international markets. Thus, rice aroma is gaining importance as a quality attribute. Therefore, basmati rice from India and Pakistan, and jasmine rice from Thailand, command higher market prices.

[0003] 2-Acetyl-1-pyrroline (2-AP) has been identified as the major aroma compound responsible for the pleasant aroma of fragrant rice. 2-AP is also responsible for the "popcorn aroma" of foods and has a very low odor threshold (0.1 μg / kg). Therefore, it can still be detected by the human nose at very low concentrations.

[0004] 2-AP was first identified as the most important flavor compound in cooked rice. However, since its discovery, 2-AP has continued to reveal its presence in a wide variety of biological systems and foods. Apart from biological systems and foods, 2-AP can also be formed in the Maillard reaction in low yields. The amino acids proline and ornithine have been discussed in the literature as precursors of 2-AP by reacting to 2-AP upon heat treatment.

[0005] The pyrroline ring of 2-AP makes the compound highly unstable. As far as commercial use is concerned, the unstable nature of 2-AP makes it unsuitable for commercial synthesis. Furthermore, natural or "clean label" versions are clearly preferred over synthetic versions. Therefore, other methods must be identified to apply the desired 2-AP aroma to foods. [Brief explanation of the drawings]

[0006] [Figure 1] Mass spectrum of 2-AP. [Figure 2] Mass spectrum of the O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine (PFBHA) derivative of 2-AP. [Figure 3] Standard curve of 2-AP using pandan leaf essence (10x). [Figure 4] Amount of 2-AP as a function of the temperature applied during the thermal process of lettuce core stems. [Figure 5] Amount of 2-AP in function of incubation duration at 100°C. [Figure 6] Solid-phase microextraction and gas chromatography coupled to mass spectrometry (SPME-GC / MS) analysis of 2-AP in seven different batches (from different parts) of lettuce before and after incubation at 100°C for 50 minutes. The peak range shows m / z 83 for the extracted ion. [Figure 7] Stability of 2-AP in the base fraction of lettuce essence. [Figure 8] Concentration of 2-AP by solid phase extraction (SPE). [Figure 9] Levels of 2-AP in the supernatant and residue of cooked lettuce. [Figure 10] Levels of 2-AP before and after incubation for both normal and reconstituted supernatants after lyophilization. [Figure 11] SPME-GC / MS analysis of white rice (top) and cooked rice (bottom) (EIC at m / z 83).

[0007] Detailed Description of the Invention The present invention relates to an extract from lettuce (Lactuca sativa) containing 2-acetyl-1-pyrroline (2-AP) and, optionally, precursors of 2-acetyl-1-pyrroline.

[0008] An extract is a preparation that contains desired compounds in a concentrated form compared to the food matrix from which they are extracted.

[0009] According to any embodiment, the extract from lettuce (Lactuca sativa) is an extract from stem lettuce (Lactuca sativa var. angustana), also known as Chinese lettuce or stem lettuce.

[0010] Preferably, the stem lettuce is a cultivar with wavy leaf blades. Even more preferably, the stem lettuce is a cultivar with wavy leaf blades and rounded tips. Even more preferably, the stem lettuce is a cultivar with wavy leaf blades, rounded tips, and long, loose internodes.

[0011] The extract of lettuce (Lactuca sativa), preferably stem lettuce (Lactuca sativa var. angustana), preferably contains precursors of 2-AP, whereby it is understood that the extract may contain precursors of 2-AP, but does not necessarily have to contain them.

[0012] 2-AP precursors are chemical compounds that can be converted into 2-AP by chemical reactions. The reaction of the precursor to 2-AP can be triggered, in particular, by heat treatment. However, UV light or the presence of any kind of catalyst can also trigger the formation of 2-AP from the precursor. The catalyst can be, for example, an acid, a base, an enzyme, or other organic / inorganic compounds, as well as mixtures thereof.

[0013] The precursors of 2-AP are reacted to 2-AP by thermal treatment at temperatures above room temperature, preferably between 30° C. and 100° C. Room temperature is defined as a temperature between 20° C. and 25° C. A conventional analytical technique for the relative and absolute quantification of 2-AP is SPME-GC / MS (solid phase microextraction and gas chromatography coupled with mass spectrometry).

[0014] In a preferred embodiment, the precursors of 2-AP are water-soluble. Compounds are considered water-soluble if more than 1 mmol of these compounds can be dissolved in 1 liter of water at 20°C.

[0015] In a preferred embodiment, the precursor of 2-AP is non-volatile. Non-volatile means that the precursor is not volatile, i.e., does not exhibit a high vapor pressure at normal room temperature. Therefore, the non-volatile precursor itself is not considered a fragrance compound.

[0016] In a preferred embodiment, the extract is obtained from lettuce leaves, peels, roots or core stalks or any mixture thereof, more preferably from lettuce leaves, peels or core stalks or any mixture thereof, even more preferably from lettuce peels or core stalks or any mixture thereof, and most preferably from lettuce core stalks. In a preferred embodiment, the lettuce leaves, peels, roots or core stalks or any mixture thereof can be used as is or can be pre-prepared, such as by grinding, before the extraction process.

[0017] In a preferred embodiment, the extract is obtained from heat-treated lettuce (Lactuca sativa), preferably stem lettuce (Lactuca sativa var. angustana), or a part thereof. The heat treatment can be carried out at a temperature selected from 25°C to 100°C, preferably 45°C to 100°C, more preferably 70°C to 100°C, and most preferably 80°C to 90°C. The heat treatment can last for a time selected from 1 to 50 minutes, preferably 30 to 50 minutes, and more preferably 40 to 50 minutes.

[0018] In a preferred embodiment, the extract is an aqueous extract or a powder extract, more preferably a powder extract. An aqueous extract means that the extracted material is entirely in solution, where the solvent comprises water. A powder extract means that the extracted material is entirely in solid, powder form.

[0019] In a preferred embodiment, the extract also contains benzaldehyde, octanal, 2-acetylpyrrole and / or nonanal, or any mixture thereof. In a more preferred embodiment, the extract also contains benzaldehyde, octanal, 2-acetylpyrrole and nonanal. Large amounts of these additional compounds can be observed in extracts obtained from heat-treated lettuce. These compounds or combinations of compounds can contribute to an even more favorable flavor impression of the extract.

[0020] The extracts of the present invention may be used as flavoring ingredients.

[0021] The present invention also relates to the use of an extract from lettuce (Lactuca sativa), preferably stem lettuce (Lactuca sativa var. angustana), containing 2-acetyl-1-pyrroline and optionally a precursor of 2-acetyl-1-pyrroline as a flavor ingredient. In other words, the present invention relates to a method for imparting, enhancing, improving or modifying the taste characteristics of a flavoring ingredient or a flavored product, which method comprises adding to the composition or product an effective amount of the extract of the present invention, for example to impart its typical notes.

[0022] A typical effective amount is 0.001 ppm to 1000 ppm, more preferably 0.1 ppm to 500 ppm, more preferably 0.5 ppm to 350 ppm, and most preferably 1 ppm to 100 ppm of the extract of the present invention based on the weight of the composition or article into which it is incorporated.

[0023] With regard to "use of the extract" it is to be understood here also the use of any composition comprising the extract of the invention which may be advantageously used in the flavour industry.

[0024] By "taste" it is meant to refer to the perception and sensation of taste.

[0025] In fact, said composition, which is advantageously used as a flavoring ingredient, is also an object of the present invention.

[0026] Therefore, the present invention provides: i. at least one extract from lettuce (Lactuca sativa), preferably stem lettuce (Lactuca sativa var. angustana), containing 2-acetyl-1-pyrroline and, optionally, a precursor of 2-acetyl-1-pyrroline, as defined above; ii. at least one ingredient selected from the group consisting of flavor carriers, flavoring co-ingredients, and mixtures thereof; and iii. optionally at least one flavor adjuvant The present invention also relates to a flavoring composition comprising:

[0027] By "flavor carrier" is meant a material that is substantially neutral from a flavor standpoint, so long as it does not significantly alter the organoleptic properties of the flavoring ingredient. The carrier may be liquid or solid.

[0028] Suitable liquid carriers include, for example, emulsifying systems, i.e., solvent and surfactant systems, or solvents commonly used in flavors. A detailed description of the nature and type of solvents commonly used in flavors is not exhaustive. Suitable solvents include, for example, propylene glycol, triacetin, caprylic / capric triglyceride (neobee®), triethyl citrate, benzyl alcohol, ethanol, vegetable oils such as linseed oil, sunflower oil or coconut oil, or terpenes.

[0029] Suitable solid carriers include, for example, absorbent gums or polymers, or even encapsulating materials. Examples of such materials include wall-forming and plasticizing materials, such as mono-, di-, or trisaccharides, natural or modified starches, hydrocolloids, cellulose derivatives, polyvinyl acetate, polyvinyl alcohol, proteins, or pectin, or further materials mentioned in references such as H. Scherz, Hydrokolloid: Stabilisatoren, Dickungs- und Geliermittel in Lebensmitteln, Band 2 der Schriftenreihe Lebensmittelchemie, Lebensmittelqualitaet, Behr's Verlag GmbH & Co., Hamburg, 1996. Encapsulation is a method well known to those skilled in the art and can be carried out using techniques such as spray drying, coagulation, extrusion, coacervation, etc.

[0030] By "flavoring ingredient" is meant herein a compound used in a flavoring preparation or composition to impart a pleasant effect. In other words, such an ingredient to be considered a flavoring ingredient must be recognized by those skilled in the art as being able to impart or modify the taste of the composition in a positive or pleasant way, and not simply having one taste.

[0031] The nature and type of flavoring co-ingredients present in the flavoring composition do not warrant a detailed description here; those skilled in the art can select them based on their general knowledge and according to the intended use or application and the desired organoleptic effect. In general terms, these flavoring co-ingredients belong to various chemical families, including alcohols, aldehydes, ketones, esters, ethers, acetates, nitriles, terpenoids, nitrogen or sulfur heterocyclic compounds, and essential oils, and the flavoring co-ingredients may be of natural or synthetic origin. Many of these co-ingredients are listed in various references, such as S. Arctander's *Perfume and Flavor Chemicals* (1969, Montclair, New Jersey, USA), or its latest edition, or other works of a similar nature, as well as in the abundant patent literature in the field of flavors. It is also understood that the co-ingredients may be compounds known to release various types of flavoring compounds in a controlled manner.

[0032] By "flavor adjuvant" is meant here an ingredient capable of imparting additional added effects, such as color, particular light resistance, chemical stability, etc. A detailed description of the nature and type of adjuvants customarily used in flavoring compositions cannot be exhaustive. Nevertheless, such adjuvants are well known to those skilled in the art who will be able to select them on the basis of their general knowledge and according to the intended use or application.

[0033] A composition consisting of at least one extract of the present invention and at least one flavor carrier represents a particular embodiment of the present invention and refers to a flavoring composition comprising at least one extract of the present invention, at least one flavor carrier, at least one flavor auxiliary ingredient, and optionally at least one flavor auxiliary.

[0034] Furthermore, the extract of the present invention can be advantageously used in the field of flavors to positively impart or improve the taste of consumer products to which said extract is added. The present invention therefore relates to flavored consumer products comprising the composition of the present invention as defined above.

[0035] The extracts of the present invention may be added to flavored consumer products, either as flavoring compositions of the present invention or as part of flavoring compositions.

[0036] For reasons of clarity, reference to a "flavored consumer product" is meant to denote an edible product, which may be a food or beverage, fried or not, and may be frozen or not, low-fat or not, marinated, battered, refrigerated, dehydrated, instant, canned, reconstituted, retorted, or preserved. Thus, an article flavored according to the present invention comprises the extract of the present invention and optional active substances, such as flavor cubes, that correspond to the taste and flavor profile of the desired edible product.

[0037] The nature and type of constituents of a food or beverage product do not warrant a more detailed description here, as those skilled in the art can select the nature and type based on their general knowledge and according to the nature of the product.

[0038] Typical examples of such flavored consumer products include: Condiments or spices, such as stocks, savory cubes, powder mixes, flavored oils, sauces (e.g., relishes, barbecue sauces, dressings, gravies, or sweet and / or sour sauces), salad dressings, or mayonnaise; Meat-based products, such as poultry, beef or pork-based products, seafood, surimi, or fish sausages; Soups, such as clear soups, cream soups, chicken or beef soups, tomato or asparagus soups; Carbohydrate-based products, such as instant noodles, rice, pasta, potato flakes or fries, noodles, pizza, tortillas, wraps; Dairy or fatty products, such as spreads, cheese, regular margarine, low-fat margarine, butter / margarine blends, butter, peanut butter, shortening, modified or flavored cheese; Flavoured products, such as snacks, biscuits (such as chips or crisps) or egg products, potato / tortilla chips, microwave popcorn, nuts, pretzels, mochi, rice crackers, etc.; Confectionery, such as bakery confectionery (e.g. sweet pastries or cakes), sugar confectionery (e.g. sweets, candy, candied nuts, chocolate, chewing gum and bubble gum, candied fruit, pastillage, sugarless confectionery or chocolate confectionery; Oral care products, such as toothpaste, mouthwash, dental care products (e.g. denture adhesives), dental rinses, mouth sprays, dental powders, dental gels or dental floss; Artificial products, such as dairy products (e.g. reformed cheese made from oils, fats and thickeners) or seafood or meat (e.g. vegetarian meat substitutes, veggie burgers) or similar products; Pet or animal food; or Beverages, such as hot drinks (e.g. tea), carbonated soft drinks, alcoholic drinks, ready-to-drink drinks or powdered soft drinks.

[0039] Some of the flavored consumer products may be aggressive media for the extracts of the present invention and may need to be protected from premature degradation, for example by encapsulation.

[0040] In a preferred embodiment, the extract is added to the food product before heat treating the food product, i.e., before cooking, roasting, or grilling. When the extract is added to the food product before heat treating the food product, the amount of 2-AP increases upon heat treatment of the food product containing the extract, because precursors of 2-AP react to 2-AP during heat treatment.

[0041] The proportion of the extract or composition of the invention that may be incorporated into the various said products varies within a wide range of values, depending on the nature of the consumer product to be flavored, as well as on the desired organoleptic effect and the nature of the auxiliary ingredients in a given base when the composition according to the invention is mixed with perfuming or flavoring ingredients, solvents or additives customarily used in the prior art.

[0042] For example, in the case of flavored consumer products, typical concentrations are from 0.001 ppm to 1000 ppm, more preferably from 0.1 ppm to 500 ppm, even more preferably from 0.5 ppm to 350 ppm, and most preferably from 1 ppm to 100 ppm of the extract or composition of the present invention by weight of the consumer product into which it is incorporated.

[0043] The present invention provides the following: a) providing lettuce (Lactuca sativa), preferably stem lettuce (Lactuca sativa var. angustana); b) isolating therefrom an extract containing 2-acetyl-1-pyrroline and, optionally, a precursor of 2-acetyl-1-pyrroline. The present invention also relates to a method for producing an extract of lettuce (Lactuca sativa), preferably stem lettuce (Lactuca sativa var. angustana), containing 2-acetyl-1-pyrroline and optionally a precursor of 2-acetyl-1-pyrroline, comprising:

[0044] The lettuce (Lactuca sativa), preferably stem lettuce (Lactuca sativa var. angustana), may be as defined above. The lettuce, in particular the leaves, skins, roots or core stalks of the lettuce as defined above, or any mixture thereof, may be provided as is or may be previously prepared, such as by crushing the lettuce before the isolation step.

[0045] The extract may be isolated by any extraction technique known in the art, such as solvent extraction or distillation, or a combination of extraction techniques.

[0046] Under solvent extraction it is to be understood the extraction of desired compounds from a food matrix with a solvent, such as water or any other suitable solvent or combination of solvents. Preferably, the solvent for extraction is an aqueous solvent, more preferably water.

[0047] Distillation defines the process of separating specific components or substances from a liquid mixture by using selective boiling and condensation. Thus, distillation exploits the differences in volatility of the components in the mixture. Various distillation techniques are well known to those skilled in the art. Steam distillation is particularly suitable for the distillation of thermally unstable aroma compounds. During steam distillation, the aroma compounds in the mixture are carried away along with the steam, making it possible to gently separate the aroma compounds from the mixture. Vacuum distillation is also particularly suitable for the distillation of thermally unstable aroma compounds. During vacuum distillation, the aroma compounds in the mixture are carried away by reduced pressure, i.e., pressure below atmospheric pressure, making it possible to gently separate the aroma compounds from the mixture in a very efficient manner.

[0048] Preferably, the extract is isolated by solvent extraction. Even more preferably, the extract is isolated by solvent extraction followed by distillation, such as steam distillation or vacuum distillation, preferably vacuum distillation.

[0049] According to a particular embodiment, method step b.) further comprises the following steps: b.1.) heat treating the lettuce to obtain processed lettuce; b.2.) steam distilling or vacuum distilling, preferably vacuum distilling, the processed lettuce of step b.1.; and b.3.) Obtaining the extract as a distillate Includes:

[0050] Heat treatment of lettuce means subjecting the lettuce to a heating step. Preferably, step b.1.) is carried out at a temperature selected from 25°C to 100°C, more preferably from 45°C to 100°C, even more preferably from 70°C to 100°C, and most preferably from 80°C to 90°C. Preferably, step b.1.) lasts for a time selected from 1 to 50 minutes, more preferably from 30 to 50 minutes.

[0051] In a preferred embodiment, the method comprises the additional step of: b.4.) acid / base extraction of the extract obtained in step b.3.), Optionally, b.5.) further concentrating the extract obtained in step b.4.), preferably by solid phase extraction, forward osmosis or pervaporation, more preferably by solid phase extraction. Includes:

[0052] Acid / base extraction is a type of liquid-liquid extraction. It typically involves different levels of solubility in water and in organic solvents. The organic solvent can be any carbon-based liquid that is insoluble in water (a non-polar solvent); common non-polar solvents are ether, ethyl acetate, dichloromethane, or pentane. Acid / base extraction can be applied to remove green aroma notes from extracts. The extract can then be acidified with an acid, such as sulfuric acid, to render compounds, such as 2-AP, ionic and therefore soluble in the aqueous phase. Thus, neutral molecules, such as many green aroma notes, can be effectively extracted with a non-polar solvent, with ionic compounds, such as 2-AP, remaining in the acidified aqueous phase. After extraction, the aqueous phase can be re-neutralized with a base, such as sodium carbonate.

[0053] Solid-phase extraction (SPE) is a sample preparation technique that separates compounds dissolved or suspended in a liquid mixture from other compounds in the mixture according to their physical and chemical properties. The compounds to be enriched are absorbed onto the solid phase, while other compounds in the sample are not. Preferably, the solid phase is octadecyl-carbon chain bonded silica (C18 phase). Thus, specific compounds in the sample can be separated from each other. The enriched compounds can then be eluted again from the solid phase with a suitable solvent (eluent). Preferably, the eluent is a solution of 70% ethanol (water / ethanol; 30 / 70; v / v).

[0054] In another particular embodiment, method step b.) comprises the steps of: b.1.) dissolving lettuce in an aqueous solution to obtain a lettuce residue and a supernatant; b.2.) Obtaining the extract as a supernatant Includes:

[0055] The aqueous solution may be any type of solvent, including water. Preferably, the aqueous solution is water. Preferably, the separation of the lettuce residue and the supernatant in step b.2.) is facilitated by centrifuging the lettuce residue and the supernatant to obtain a clear extract as the supernatant. Preferably, the separation may be performed by centrifuging the lettuce residue and the supernatant in step b.2.), followed by filtering the supernatant using membrane filtration, such as filtration, microfiltration, ultrafiltration, forward osmosis, reverse osmosis, or a combination thereof.

[0056] In a preferred embodiment, the method comprises the additional step of: c1) drying the extract, preferably by spray drying or freeze drying, or c2) Diluting the extract Includes:

[0057] Drying the extract means that essentially no solvent remains in the extract after the drying step, preferably no solvent at all. The extract is therefore solid and is powdered after the drying step. Spray drying is a method of producing a dry powder from a liquid or slurry by rapidly drying with hot gases. Freeze drying, on the other hand, is a low-temperature dehydration process in which the product is frozen, the pressure is reduced, and the ice is removed by sublimation. Both of the mentioned drying methods are gentle drying methods and are therefore particularly suitable for heat-labile compounds, such as many aroma compounds.

[0058] The extract according to the invention is preferably obtained according to the preparation method defined above.

[0059] Example Example 1: Sample preparation As samples, various parts of stem lettuce (Lactuca sativa var. angustana) are used: the heart stem, leaves, roots, and the milky fluid secreted upon wounding of the skin, and the skin (containing the milky fluid).

[0060] All fresh lettuce samples were ground to a fine powder in a cryogenic mill cooled with liquid nitrogen to ensure homogeneity. The powder samples were stored in a freezer at -80°C.

[0061] Example 2: Analysis method of 2-AP a) SPME-GC / MS 1-2 g of sample from Example 1 was placed in a 20 mL headspace vial for sampling. For SPME sampling, a Supelco DVB / CAR / PDMS SPME fiber (1 cm, 50 / 30 μm film thickness, Supelco, Bellefonte, PA) was used. Prior to sampling, each sample was equilibrated at 40°C for 10 minutes. After equilibration, the SPME fiber was exposed to the vial's headspace for 15 minutes at the same temperature (40°C). The entire sampling procedure was performed using a Gerstel autosampler for SPME. The fiber was then desorbed into a GC / MS injection port at 250°C for 3 minutes.

[0062] The GC / MS system was equipped with an Agilent 6890N GC, an Agilent 5975 mass spectrometer, and a 30 m x 0.25 mm id x 0.25 μm DB-1ms (J&W 122-0132) column.

[0063] GC / MS method: Furnace: 50°C (hold for 3 minutes) to 250°C at 100°C / min, hold for 3 minutes. Inlet temperature: 250°C. Carrier gas: helium. Flow rate: 0.7 mL / min. Split ratio: 25:1. Mass spectrometer ionization voltage: 70 eV. Scan range: m / z 29-450. Quadrupole temperature: 150°C. Ion source temperature: 230°C.

[0064] 2-AP is detected on a DB-1ms column at an LRI of 886 and in the mass spectrum as shown in FIG.

[0065] b) Derivatization and quantification of 2-AP with PFBHA An excess amount of PFBHA (O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine) was added to an aqueous sample containing 2-AP and mixed properly. The reaction was allowed to stand at room temperature for 2 hours, after which the reaction was extracted with ethyl acetate (EtOAc) and the organic phase was injected into a GC / MS. The linear retention index (LRI) value of the PFBHA derivative of 2-AP in a DB-1ms column was 1625, and its mass spectrum is shown in Figure 2.

[0066] The GC / MS system was equipped with an Agilent 6890N GC, an Agilent 5975 mass spectrometer, and a 30 m x 0.25 mm id x 0.25 μm DB-1ms (J&W 122-0132) column.

[0067] GC / MS method: Furnace: 50°C (5 min hold) to 300°C at 5°C / min, then 340°C at 50°C / min (3 min hold). Inlet temperature: 250°C. Carrier gas: helium. Flow rate: 0.7 mL / min. Split ratio: 25:1. Mass spectrometer ionization voltage: 70 eV. Scan range: m / z 29-450. Quadrupole temperature: 150°C. Ion source temperature: 230°C.

[0068] For lettuce samples with insoluble components, the samples are first sonicated at 45°C for 30 minutes and filtered. Excess PFBHA is added to the filtrate and mixed thoroughly. The reaction is left at room temperature for 2 hours, after which the reaction is extracted with ethyl acetate (EtOAc) and the organic phase is injected into a GC / MS.

[0069] Using this method, a calibration curve of 2-AP was established with pandan leaf essence (10x).

[0070] c) Quantification of 2-AP with internal standard Quantification is carried out using vanillin as internal standard according to the procedure described in method b).

[0071] Example 3: Detection of 2-AP in various lettuce parts The various lettuce parts (heart stem, leaves, roots, and the milky fluid secreted upon wounding of the skin, and the skin (containing the milky fluid)) were separated from a single lettuce plant, and the mass of each part is shown in Table 1. Each part was analyzed by the SPME-GC / MS method. Except for the milky fluid (0.1 g), all other parts were analyzed in 2-gram samples. The results are shown in Table 1. The heart stem, which is the heaviest part of the lettuce, shows the highest levels of 2-AP.

[0072] [Table 1]

[0073] Example 4: Significant increase of 2-AP in cooked lettuce One gram of powdered lettuce heart stem (-80°C) was placed in a 20 mL headspace sampling vial. Several vials were prepared in this manner and incubated at different temperatures (25°C, 45°C, 70°C, and 100°C) for 30 minutes. After incubation, all samples were cooled to room temperature and analyzed by SPME-GC / MS (Figure 4). 2-AP levels increased significantly with temperature. Temperatures of 100°C, the limit of typical cooking conditions, were never exceeded.

[0074] Example 5: Optimization of incubation time The incubation time was further optimized at 100°C. One gram of powdered lettuce heart stems (-80°C) was placed in a 20 mL headspace sampling vial. Several vials were prepared in this manner and incubated at 100°C for different times (10, 20, 30, 40, and 50 minutes). The samples were cooled to room temperature and analyzed by SPME-GC / MS (Figure 5). Clearly, 40 to 50 minutes of incubation at 100°C was the optimal time to produce the highest levels of 2-AP.

[0075] Example 6: Thermogenesis of 2-AP in different parts and different batches of lettuce Seven different batches of lettuce were analyzed by site (leaf, peel, and core) for 2-AP using SPME-GC / MS before and after incubation at 100° C. for 50 minutes. The results are shown in FIG.

[0076] Example 7: Preparation of essence from cooked lettuce 40 g of powdered lettuce heart stalks were placed in a glass vial, tightly sealed, and incubated at 100°C for 1 hour. After cooling to room temperature, the sample was transferred to a flask for distillation. Distillation was carried out in a Solvent-Assisted Flavor Extraction apparatus (SAFE) at 65°C, and the vacuum was adjusted to bring the sample to a slight boil. Distillation was completed in 0.5 hours, and the distillate was collected as lettuce stem aroma water. The aroma water had a typical aroma of lettuce stems (green + aromatic rice). Further SPME-GC / MS analysis of the aroma water indicated that the green smell was mainly derived from C6 alcohols and aldehydes.

[0077] Example 8: Preparation of the base fraction of the essence The essence of Example 7 was further treated by acid / base extraction to remove the green notes from the essence.

[0078] The aroma water was first acidified to pH = 3 with 10% sulfuric acid and washed three times with pentane to remove neutral molecules. The acidic water was collected and neutralized to pH 8 with Na2CO3. SPME-GC / MS analysis of this base fraction showed a volatile profile predominantly consisting of 2-AP.

[0079] Example 9: Stability of 2-AP in the base fraction of the essence The basal compartment was tightly closed and placed at room temperature. Control samples were frozen at -80°C. Samples at room temperature and -80°C were analyzed at different time points (days 0, 19, 52, and 84). Minor degradation of 2-AP was observed on day 52, and significant degradation was observed on day 84 (Figure 7).

[0080] Example 10: Preparation of concentrated essence by solid phase extraction (SPE) The lettuce essence of Example 8 was further concentrated by SPE. 200 g of the basic fraction of the lettuce essence prepared according to the procedure described in Example 8 was loaded onto a C18 column (1 g). The column was then eluted three times with 1 mL of 70% ethanol. Each fraction (100 μL) was diluted 10-fold with water to 1 mL and analyzed by SPME-GC / MS. Fraction 2 showed the highest level of 2-AP, approximately 15 times the concentration of the original essence ( FIG. 8 ). Both Fractions 1 and 2 had a strong, clean rice aroma.

[0081] Example 11: Precursor Studies To demonstrate the effect of 2-AP precursors on 2-AP formation during heat treatment, lettuce heart stalk slurries (powdered samples warmed to room temperature) were centrifuged to separate the supernatant and residue. The residue was further washed three times with water. Four samples were prepared in 20 mL headspace sampling vials as follows: Sample 1: 1 g of supernatant; Sample 2: 0.5g supernatant + 0.5g residue; Sample 3: 0.5g supernatant + 0.5g water; Sample 4: 1g residue.

[0082] All samples were incubated at 100°C for 50 minutes, and the 2-AP content was measured by derivatization (Fig. 9).

[0083] Sample 1 showed twice the level of 2-AP as in samples 2 and 3, while sample 4 had very low levels of 2-AP, indicating that all the precursors required for 2-AP production in the thermal process were present in the supernatant and that the residue contained only a small amount of precursor.

[0084] Ten grams of the supernatant was freeze-dried for an additional two days to remove as much water and volatiles as possible. The freeze-dried residue was reconstituted to 10 grams with water. The reconstituted and normal supernatants were analyzed for 2-AP by SPME-GC / MS before and after incubation at 100°C for 50 minutes.

[0085] Analysis showed that the reconstituted supernatants exhibited slightly more 2-AP than the regular supernatants both before and after incubation (Fig. 10), indicating that all precursors required for 2-AP production upon heat treatment were minor volatile components.

[0086] Example 12: Rice cooked with freeze-dried aqueous lettuce extract To test the pro-flavor properties of the freeze-dried aqueous lettuce extract, 60 g of lettuce stalk supernatant was freeze-dried for 24 hours, yielding 3.2 g of a light green powder. 100 g of regular rice was cooked with 1.6 g of the freeze-dried powder in a rice cooker. A control sample was cooked without the addition of lettuce extract. Both samples were cooled to room temperature and evaluated (chewed and swallowed) by 12 panelists; 11 tasted a significantly stronger rice aroma, and 3 found the sample cooked with lettuce extract to be sweeter.

[0087] Two samples (2 g) were also analyzed by SPME-GC / MS. 2-AP was observed in the sample cooked with lettuce extract, but not in the control sample (Figure 11).

[0088] Example 13: Lettuce powder The following procedure was performed on 20.3 kg of fresh lettuce.

[0089] aperture Fresh lettuce was squeezed twice in a juicer to obtain 14.4 kg of juice.

[0090] filtration The juice was fed to a microfiltration plant with a membrane size of 0.1 μm. After filtration, 8.7 kg of filtrate was obtained.

[0091] spray drying 0.06 kg of carrier, capsules and 0.56 kg of maltodextrin 18DE were added to the filtrate and mixed for 30 minutes. The solution was introduced into a spray dryer at a flow rate of 400 ml / hr. 0.6 kg of product was obtained.

Claims

1. An extract from lettuce (Lactuca sativa) containing 2-acetyl-1-pyrroline and, optionally, a precursor of 2-acetyl-1-pyrroline.

2. The extract according to claim 1, wherein the lettuce (Lactuca sativa) is stem lettuce (Lactuca sativa var. angustana).

3. 3. The extract according to claim 1 or 2, wherein the extract is obtained from lettuce leaves, peels, roots or core stalks or any mixture thereof, preferably the extract is obtained from lettuce leaves, peels or core stalks or any mixture thereof, even more preferably the extract is obtained from lettuce peels or core stalks or any mixture thereof, most preferably the extract is obtained from lettuce core stalks.

4. 4. The extract according to any one of claims 1 to 3, wherein the extract is an aqueous extract or a powder extract.

5. below, i. At least one extract from lettuce (Lactuca sativa), preferably stem lettuce (Lactuca sativa var. angustana), containing 2-acetyl-1-pyrroline and, optionally, a precursor of 2-acetyl-1-pyrroline. ii. at least one ingredient selected from the group consisting of flavor carriers, flavoring co-ingredients, and mixtures thereof; and iii. Optionally, at least one flavor adjuvant A flavoring composition comprising:

6. A flavored consumer product comprising an extract according to any one of claims 1 to 4 or a composition according to claim 5.

7. Use of an extract from lettuce (Lactuca sativa), preferably stem lettuce (Lactuca sativa var. angustana), containing 2-acetyl-1-pyrroline and optionally a precursor of 2-acetyl-1-pyrroline as a flavoring component.

8. 1. A method for imparting, enhancing, improving or modifying the taste characteristics of a flavoring composition or flavored article, the method comprising adding to said composition or article an effective amount of an extract from lettuce (Lactuca sativa), preferably stem lettuce (Lactuca sativa var. angustana), comprising 2-acetyl-1-pyrroline and optionally a precursor of 2-acetyl-1-pyrroline.

9. below, a) providing lettuce (Lactuca sativa), preferably stem lettuce (Lactuca sativa var. angustana); b) isolating therefrom an extract containing 2-acetyl-1-pyrroline and, optionally, a precursor of 2-acetyl-1-pyrroline.

1. A method for producing an extract of lettuce (Lactuca sativa), preferably stem lettuce (Lactuca sativa var. angustana), containing 2-acetyl-1-pyrroline and optionally a precursor of 2-acetyl-1-pyrroline, comprising:

10. Step b) comprises the following steps: b.1.) heat treating lettuce to obtain processed lettuce; b.2.) steam distilling or vacuum distilling, preferably vacuum distilling, the processed lettuce of step b.1.; and b.3.) Obtaining the extract as a distillate 10. The method of claim 9, comprising:

11. 11. The process according to claim 10, wherein step b.1.) is carried out at a temperature selected from 25°C to 100°C, preferably 45°C to 100°C, more preferably 70°C to 100°C, and most preferably 80°C to 90°C.

12. The method according to claim 10 or 11, wherein step b.1.) lasts for a time selected from the range of 1 to 50 minutes, preferably 30 to 50 minutes.

13. Next additional step: b.4.) Acid / base extraction of the extract obtained in step b.3.), Optionally, b.5.) further concentrating the extract obtained in step b.4.), preferably by solid phase extraction.

13. The method of any one of claims 10 to 12, comprising:

14. Step b) comprises the following steps: b.1.) Dissolving lettuce in an aqueous solution to obtain a lettuce residue and a supernatant; b.2.) Obtaining the extract as a supernatant 10. The method of claim 9, comprising:

15. Next steps: c) drying the extract, preferably by spray drying or freeze drying 15. The method of any one of claims 9 to 14, comprising:

Images