"Cooling" Compounds Formed from Carbohydrates ... - ACS Publications

Chapter 25

Activity-Guided Screening and Identification of Natural "Cooling" Compounds Formed from Carbohydrates and L-proline in Beer Malt T. Hofmann, A. Bareth, and H. Ottinger

Downloaded by CORNELL UNIV on August 26, 2016 | http://pubs.acs.org Publication Date: December 1, 2003 | doi: 10.1021/bk-2004-0871.ch025

Deutsche Forschungsanstalt für Lebensmittelchemie, Lichtenbergstrasse 4, D-85748 Garching, Germany

Gel permeation chromatography (GPC) of the solvent-extractables isolated from a thermally treated glucose/L-proline mixture and sensory analysis of the fractions collected led to the discovery of the presence of "cooling" compounds in Maillard reactions. To characterize the compounds imparting this oral cooling sensation, the Taste Dilution Analysis was applied to the cooling-active GPC fraction by determining the taste threshold of reaction products in serial dilutions of HPLC fractions. MS, NMR, [ C] labeling experiments, followed by synthesis led to unequivocal identification of 3-methyl-2-(1-pyrrolidinyl)-2-cyclopenten-1-one (3-MPC), 5-methyl-2(1-pyrrolidinyl)-2-cyclopenten-1-one (5MPC) and 2,5-dimethyl-4-(1-pyrrolidinyl)-3(2H)-furanone (3(2H)DMPF) as the most intense "cooling" compounds formed from hexoses. Comparative studies on pentose/L-proline mixtures led to the identification of the odorless 5-methyl-4-(1-pyrrolidinyl)3(2H)-furanone (3(2H)-MPF), exhibiting a "cooling" sensation at low concentrations of 1.5-3.0 mg/kg (water), as one of the most active "cooling" agents reported so far. To the best of our knowledge, these are the first Maillard reaction products reported to cause intense cooling sensations by degustation. Finally, the detection of 5-MPC, 3-MPC, 3(2H)-DMPF, and 3(2H)-MPF in dark roasted beer malts verified their natural occurence in thermally processed foods and demonstrated 3(2H)-MPF as the most active, odorless cooling agent reported so far in nature. 13

Malt is produced by steeping and germination of cereals, in particular barley, followed by a kiln-drying and/or a roasting process. Depending on the processing parameters during malt manufacturing, a variety of malt with different aroma, taste and color attributes are obtained. Thermal reactions, in the particular Maillard reaction between reducing carbohydrates and amino acids liberated from corresponding biopolymers during germination, are chiefly responsible for the development of the unique aroma and taste as well as the typical brown color of 338

© 2004 American Chemical Society Shahidi and Weerasinghe; Nutraceutical Beverages ACS Symposium Series; American Chemical Society: Washington, DC, 2003.


339 malt. Although not used as a food as such, malt is used for centuries in the manufacturing of whiskey and beer, and gain growing interest as flavoring and coloring material in, e.g. breadmaking or the manufacturing of breakfast cereals. Although the consumer acceptance of these foods is strongly influenced by a balanced interplay between the aroma-active (perceived in the nose) as well as the taste-active components (detected on the tongue), the information available on sapid taste compounds present in dark malt is as yet very limited. Diketopiperazines, such as, e.g. cyclo(Phe-Pro), cyclo(Leu-Pro), cyclo(Pro-Pro), cyclo(Val-Pro) and cyclo(Ile-Pro), which are formed by dimerization of amino acids or peptides, have been reported as bitter tastants in roasted malt (7). To answer the question whether besides such diketopiperazines taste compounds are also formed from carbohydrates and amino acids during malt manufacturing, numerous studies have been focused on the identification of sensorially active reaction products formed in food-related, Maillard-type model mixtures. Because steeping and germination of barley produce L-proline and glucose as the quantitatively predominating Maillard precursors prior to the kilning and roasting process, the reaction between these components has been extensively investigated in the last decades to model the flavor formation during the manufacturing of roasted malts. Because most of these identification experiments have been primarily focused on the quantitatively predominating products formed, rather than selecting the target compounds with regard to taste-activity, only a few taste compounds have been identified so far, e.g. pyrrolidino-hexose-reductones {2-4), pyrrolidines (5) and cyclopent[o]azepin-8-ones (5-7). In order to bridge the gap between pure structural chemistry and human taste perception, a more straightforward technique, the so-called Taste Dilution Analysis (TDA) has recently been developed, which is based on the determination of the detection threshold of taste compounds in serial dilutions of HPLC fractions (#). This novel bioassay offers the possibility to rank food components according to their relative taste impact and has proved to be a powerful technique for the identification of key taste compounds; e.g. the novel 3-(2-furyl)-8-[(2-furyl)methyl]-4-hydroxymethyl-l-oxo-l/f,4^-quinolizinium-7olate, exhibiting an extraordinarily low detection threshold of 0.00025 mmol/kg water, was successfully identified as the most intense bitter tasting compound formed during thermal treatment of pentoses and primary amino acids {8). Until now, all the taste-active compounds detected in Maillard reaction mixtures or in roasted malts showed bitter taste qualities only. However, no information is available on the chemical structures of more desirable tastants. The aim of the present investigation was, therefore, to identify taste compounds exhibiting desirable sensory qualities in heated carbohydrate/L-proline mixtures and to verify their natural occurrence in processed cereals, i.e. in dark malt.

Experimental Materials The following compounds were synthesized as following the procedures reported recently: 2,4-dihydroxy-2,5-dimethyl-3(2//)-furanone (P), 3-deoxy-2hexosulose {10), 3,5-dihydroxy-2-methyl-5,6-dihydropyran-4-one {11), 3-MPC {12), 5-MPC (72), 3(27/)-DMPF (72), and 3(277)-MPF (75). Malts were supplied by the German brewing industry. Shahidi and Weerasinghe; Nutraceutical Beverages ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

340 Thermally treated carbohydrate/L-proline mixture Glucose (200 mmol) or xylose (200 mmol), respectively, was dry-heated in the presence of L-proline (200 mmol) for 20 min at 190°C, cooled to room temperature, suspended in hot water (1.5 L) andfiltered.The solvent extractable reaction products were isolated with CH C1 and separated by gel permeation chromatography. 2

2


Gel permeation chromatography (GPC) The solvent extractables of the glucose/L-proline mixture (2.89 g) were separated by gel permeation chromatography on Sephadex LH-20 (750 mm χ 55 mm, Pharmacia, Upsala, Sweden) using a mixture (75/25, v/v; 3 mL/min) of methanol and aqueous ammonium formate (50 mmol/L; pH 3.5) as the eluent (12). Monitoring the effluent at 300 nm, ten fractions (Figure 1) were collected, freed from solvent at 30°C in vacuo (45 mbar), and then freeze-dried. The material of eachfractionwas dissolved in tap water and used for sensory analysis (Table I). Thermally treated mixtures of hexose-derived intermediates and L-proline Mixtures of L-proline (10 mmol), the hexose intermediates (10 mmol) given in Table II, and A1 0 (10 g, neutral) were dry-heated for 10 min at 180°C. After cooling, the mixtures were taken up in hot water (500 mL), and the solvent extractables were analyzed by HPLC/taste dilution analyses (12). 2

3

HPLC/taste dilution analysis (HPLC/TDA) An aliquot (200 mg) offractionV (Figure 1), the solvent-extractable fraction of the cyclotene/L-proline mixture or the 2,5-dimethyl-4-hydroxy-3(2/f)furanone/ L-proline mixture (data not given), respectively, was dissolved in methanol, and analyzed by RP-HPLC. The effluent was separated into 30 (GPC fraction V; Figure 2) or 16 fractions (cyclotene/L-proline mixture; Figure 4), respectively, on which the HPLC/taste dilution analysis was applied as recently reported (12). 12

13

[ C/ C] Labeling experiment !3

Glucose (0.5 mmol), [ C ] glucose (0.5 mmol) and L-proline (1.0 mmol) were mixed and dry-heated for 15 min at 190°C. After solvent extraction and column chromatography, HRGC/MS revealed the isotopomeric patterns of the molecular ions of the "cooling" compounds detected by HPLC/TDA of fraction V (12). 6

Identification of "cooling" compounds in dark malts Ground malt (50 g) was stirred overnight with dichloromethane (2 χ 400 mL), the combined organic layers were concentrated to about 50 mL, and the volatile components were isolated by high vacuum distillation at 35°C (14). After pre-separation by column chromatography on A1 0 , 3-MPC, 5-MPC, 3(2H)DMPF, and 3(2//)-MPF were identified and quantified by comparison of the 2

3

Shahidi and Weerasinghe; Nutraceutical Beverages ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

341

retention times, the mass spectra as well as the peak areas with those obtained for the synthetic reference compounds (72).

Results and Discussion


HPLC analysis of the solvent-extractable fraction isolated from a roasted equimolar mixture of glucose and L-proline showed that a tremendous multiplicity of reaction products had been formed. To sort out the strongly tasteactive compounds from the bulk of less taste-active or tasteless substances, the reaction products were separated from the high-molecular, melanoidin-type material by means of gel permeation chromatography on Sephadex LH-20. Monitoring the effluent at 300 nm, the GPC chromatogram, displayed in Figure 1, was recorded and tenfractions(Fractions I to X) were collected separately.

-,— 2

ι

1

1

r

4

6

8

10

t(H)

Figure J. UfL chromatogram of the solvent-extractable jractwn of a dryheated glucose/L-proline mixture In order to evaluate their taste activity, these GPCfractionswere freezedried, the residues were taken up in tap water and then presented to a trained sensory panel which was asked to judge the taste qualities of thesefractionsby gustation in a triangle test (72). Whereas fraction I andfractionsVIII-X did not show any taste impact,fractionsII to VI tasted bitter (Table I). It was, however, interesting to notice thatfractionV, showing the most intense absorption at 300 nm (Figure 1), in addition to the bitter taste, imparted a significant "cooling" effect to the tongue of the panelists (72). Because the presence of "cooling" compounds in Maillard reactions has not previously been reported, the following identification experiments focused onfractionV. Shahidi and Weerasinghe; Nutraceutical Beverages ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

342 Table I. Taste qualities and yields of fractions obtained by GPC of the solvent-extractable reaction products formed from glucose and L-proline yield

a


fraction no.

I II III IV V VI VII VIII IX X

taste quality

b

[mg]

[%]

202 428 720 855 415 120 53 28 10 ' II

3(2if)-MPF

Figure 9. Reaction pathways leading to theformation ofthe "cooling "-active 5-methyl-4-(l^ynolidinyl)-3(2H)-furanone(3(2H)-MPF) Identification of "cooling" Maillard compounds in dark malts In order to prove the "naturalness" of these "cooling" Maillard compounds, a solvent-extractable, volatile fraction was isolated from ground malts, and, after preseparation by column chromatography, was analyzed for 3-MPC, 5-MPC, 3(2#)-DMPF and 3(2#)-MPF by meanse of HRGC/MS (72,16). As examplified for 3-MPC and 5-MPC in Figure 10, all four "cooling" compounds could be identified in dark roasted malts by comparison of the retention times as well as mass spectra (EI, CI) with those obtained for the synthetic reference compounds. The highest concentrations of the "cooling" compounds were found in caraffa malt (Table III), which is a dark roasted speciality malt, e.g. 101.3 μg 5-MPC were present per kg caraffa malt (72). In comparison, a melanoidin malt contained by factors of 4 to 7 lower amounts of these Maillard compounds, e.g. 13.5 μg/kg 5-MPC were determined, which is well in line with the lower roasting degree compared to the caraffa malt. The low degree of Maillard reactions in the light kiln-dried Pilsener malt did not generate the "cooling" compounds in significant amounts. Table III. Concentrations of "cooling" Maillard compounds in malts compound Pilsener malt 5-MPC 3-MPC 3(2//)-DMPF 3(277)-MPF

"Cooling" Compounds Formed from Carbohydrates ... - ACS Publications

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