formation of maltosine, a product of the maillard reaction with a pyridone structure

Original papers Originalarbeiten

Formation of maltosine, a product of the Maillard reaction with a pyridone structure

Franz Ledl, Helga Osiander, Otto Pachmayr, and Theodor Severin Institut fiir Pharmazie und Lebensmittelchemie der Universitfit Miinchen, Sophienstrasse 10, D-8000 Mfinchen 2, Federal Republic of Germany

Bildung von Maltosin: Ein Produkt der Maillard-Reaktion mit Pyridonstruktur

Zusammenfassung. Beim Erhitzen von N~-Acetyllysin mit Maltose oder Lactose bildet sich u.a. das bisher nicht bekannte Pyridon 8 aus dem nach Abspaltung der Acetylgruppe das Lysinderivat 9 erhalten wird. Ffir die neue Substanz 9 schlagen wit die Bezeichnung Maltosin vor. Im Gegensatz zum Maltosin ist das schon fr/iher bekannte und zum Nachweis einer Ly- sinseh/idigung herangezogene Pyridosin ein ,,Kunst- produkt" der sauren Proteinhydrolyse. Aus den Um- setzungsgemisehen von Galaktosylisomaltol und eines Amadoriprodukts der Maltose mit Propylamin lassen sich die Pyridiniumbetaine 13 und 1~ isolieren. Sic sind Zwischenprodukte bei der Bildung des Pyridons 12a.

Summary. By heating N'-acetyllysine with maltose or lactose, the pyridone 8_ not known up to now is formed, which is transformed into the lysine derivative 9 when the acetyl group is split off. We propose the name maltosine for compound _9. In contrast to maltosine, the well-known pyridosine, used in the deter- mination of lysine damage, is an "artificial" product formed during acid protein hydrolysis. From the reaction mixtures of galactosyl isomaltol and an Amadori product of maltose with propylamine, the pyridinium betaines 13 and 14 can be isolated. They are intermediates in the formation of the pyridone 12a.

Introduction

The first steps in the Maillard reaction, i.e. the conversion of reducing sugars with amino acids, have been known for a long time and have been investigated in detail by various research groups. For example, glucose can be readily added to amino acids to form cor-

Offprint requests to." F. Ledl and T. Severin

responding N-substituted glyeosylamines, which are converted into "fructose amino acids" via an Amadori rearrangement [1]. Accordingly, lactose in heated milk reacts with the amino groups of the lysine side-chain of milk proteins to form protein-bound lactulose lysine. If such a protein is subjected to acid hydrolysis, the lysine derivatives furosine [2] and pyridosine [3] are found in the chromatogram in addition to the usual amino acids. Initially, these heterocycles are not present in heat-damaged proteins and are only formed by the action of hot hydrochloric acid from the Ama- dori compounds under the conditions usual for protein cleavage. The structures o f ! and 2 have been veri- fied by detailed investigations. Particular attention has been paid to pyridosine and furosine in food analysis. The formation of Amadori compounds and the heat damage of proteins can be demonstrated via these substances [4]. In this respect, it is of particular interest that fructose lysine cannot be utilized in the digestive tract according to the results of rat feeding trials [5].

In our investigations on Maillard reactions, we have also heated maltose and lactose with methylam- monium acetate and glycine in water and analysed the components which can be extracted with methylene chloride or acetyl acetate. We obtained cristalline 1,2- dimethyl-3-hydroxy-pyridone 3_Aa and 1-carboxy- methyl-2-methyl-3-hydroxy-pyridone 3b in relatively large amounts [6]. The same pyridones are not formed or are only formed in a small amount from monosac-

. . . . . . HN-CH-CO . . . . . . . ~ ' ~ I CO-CH z

(? H2)~ NH NH 1 [ I = {CH~)4

I CHz 6N HCI H2N- CH-COOH C=O 112°C 2Z, h O I

HO-CHI HO'~J.i

HC-OR CH 3 i P

HC- OH i (CH2)'~ HzC- OH HzN_CH_COOH

R = H, o(-glu,13-gal 2 =

Fig. 1

Z Lebensm Unters Forsch (1989) 188:207-211 © Springer-Verlag 1989

~H

maltose, lactose

Fig. 2

0

I CH~ HzN-CH-COOH

4 =

Fig. 3

* NHzR OH

CH 3

R

3 a: R=CH 3 =

b : R = CHz-COOH

0 ~]~ OH l ~ O - 1 3 - - g a i

CH 3 " 0 "COCH 3

5 6

charides such as glucose and fructose. On the basis of these results, it can be expected that pyridones are also formed by heating disaccharides with proteins. The amino group of the lysine side-chain should be incor- porated preferentially into the heterocycle. In order to check this, we have converted N%acetyl lysine with various reducing sugars.

Among the products of the Maillard reaction, 3- hydroxypyridones are of special interest. Kon- toghiorghes and Evans [7] found such compounds to be strong chelating agents for trivalent metals. Thus, the 1,2-dimethyl-3-hydroxy-pyridone 3a removes iron completely from ferredoxin [7]. Aluminium is also strongly bound in a complex by3a [8]. Mimosine (4) a pyridone with amino acid side-chain was recently isolated from Leguminosae species [9]. The high toxic- ity of the compound is interesting. Besides general de- bility and infertility, teratogenic effects were also ob- served, e.g. in rats and pigs. The structure ofpyridones resembles that of maltol (5), whose structure has been known for a long time [10]. This is a substance which is formed in the thermal decomposition of disaccha- tides and polysaccharides, but hardly from monosac- charides. Galactosyl isomaltol 6, which has only recently been investigated in detail, is formed in large quantities from lactose l11].

Experimental section

General methods

Melting points (mp, uncorrected) were determined with a Biichi 510 apparatus. IR spectra were recorded in KBr discs with a Perkin- Elmer 197 spectrometer. NMR spectra (internal standard; tetra- methylsilane) were recorded with a Varian A-60 spectrometer at 60 MHz and a Jeol GSX-270 instrument at 270 MHz. Mass spectra were obtained with a Varian MAT CH7 spectrometer equipped with a probe inlet at 70 eV and a Kratos MS-80 instrument.

Silica gel 60Fzs 4 (2 mm, Merck, 57•7 and 0.5 mm, Merck, 5744) was used for thin layer chromatography (TLC) and silica gel 60 (Merck, 9385) for column chromatography. Low-pressure chromatography was achieved with a reversed-phase (RP)-18 column

(Merck, 10625, 40-60 gm, type B) and a silica gel column (Merck, 10625, 40-63 gm, type B). High-performed liquid chromatography (HPLC) was performed on a Merck-Hitachi system (pump L-6000, UV detector L-4000 and chromato-integrator D-2000) with LiChrosorb RP-18 columns (Merck, 50994, 7 gm, 250 x 10 mm i.d.; Bischoff, 25461855, 5 gm, 250 x 4.6 mm i.d.).

Gaschromatography (GC) was performed with a Perkin-Elmer 8320 instrument equipped with a flame-ionization detector; injection and detection ports at 260 °C; GC system 1: quartz capillary column (25 m x 0.25 mm, coated with CPMS 1701) with temperature pro- gram 180°-260°C at 6°C/min; GC system 2: quartz capillary column (25 m x 0.3 mm, coated with DMS 025) with temperature pro- gram 100°-260 °C at 10 °C/min. Ion-exchange chromatography was performed with an LKB 4400 instrument (steel columns 4 x 295 mm, filled with Ultro Pac-8; temperatures: tl = 45 °C, G = 56 °C, and G = 73 °C; sodium citrate buffer 0,2 mol/l: e~ = pH 3.20, ez = 4.25, and e3 = 6.45 with the addition 1.0 moll1 sodium chloride; detection was carried at 440 and 570 nm after the addition of ninhydrine. El- ementary analysis was performed with a Rapid Heraeus instrument.

Synthesis of aeetyl maltosine 8

N'-Acetyl lysine [12] (5,7 g 0.03 tool) and isomaltol (7) [13] (5 g, 0.04 tool) were heated in water (60 ml) for 20 h under reflux. Unreacted isomaltol was removed with methylene chloride (7 h) and compound 8 was extracted with ethyl acetate (9 h). The organic layer was dried over sodium sulphate and concentrated under diminished pressure. To the syrupy residue, a small amount of methanol was added and undissolved N~-acetyl lysine was removed by filtration. Column chromatography of the filtrate on silica gel with ethyl acetate and increasing amounts of methanol gave a fraction containing 8 (TLC control, with developing solvent ethyl acetate/methanol (3 + 7) and spray reagent iron-III-chloride). The solution solvents were removed under diminished pressure and _8 was obtained as a colourless, hygro- scopic oil (3.5 g, 40%). The spectral data for 8 are as follow: 1H- NMR (CD3OD): 6 1.2-2.3 (m, 6H), 2.04 (s, 3H),2.51 (s, 3H), 3.7-4.8 (m, 3H), 6.53 (d, IH), 7.73 (d, 1H). MS: m/z 296 (23%, M.+), 278 (70), 207 (92), 180 (80), 166 (60), 164 (32), 152 (58), 126 (53), 125 (100), 96 (81), 84 (67). - IR (KBr): 3280, 2950, 1720, 1650, 1630, 1550, 1500, 1370, 1260, 1220, 1040, 830 cm-1 ._ UV (CH3OH): 2m~x = 286 nm (lge = 4.09). The elemental analysis calculated for C, H, N are: C, 56.75%; H, 6.80%; N, 9.45%; the percentage actually found were: C, 56.37%;H, 7.44%; N, 9.19%.

Isolation of acetyl maltosine 8from reaction mixtures of disaccharides and N~-acetyl lysine

A 7.2 g sample (0.02 mol) of the disaccharide (maltose, lactose) and N~-acetyl lysine (3.8 g, 0.02 moo were dissolved in water (50 ml, pH 7, phosphate buffer) and heated for 24 h under reflux. After cooling, the phosphate buffer was partly precipitated by the addition of methanol. The filtrate was concentrated under reduced pressure and the residue was fractionated on silica gel (conditions as described for the synthesis of_8). The syrupy residue of the fractions, in which _8 was detected, was purified with low-pressure chromatography (silica gel) starting with ethyl acetate/methanol (1 +1), followed by mixtures with increasing amounts of methanol. In a fraction eluated with ethyl acetate/methanol (1 + 7), 8 was identified (TLC control with the au- thentic synthesized compound). Further purification of the residue was performed by HPLC (7 pm) with water/acetonitrile (20+1) (flow rate 2 ml/min, R t = 14.07 min) and again by HPLC (5 gm) with triethylammonium formiate (0.02 mol, pH 4)/acetonitrile (85 + 15) (flow rate 1 ml/min, R t = 4.32 min). After repeated injec- tions and collection of the eluate at 4.32 min, a sufficient amount of 8_ was obtained to get spectral. They were identical to those obtained from synthesized acetyl maltosine 8_.

208

Ion exchange chromatography of maltosine (9._) andpyridosine (2_)

Maltosin (9) was obtained by hydrolysis of acetyl maltosine 8 (95 mg, 5 ml of 6 N-hydrogen chloride, 24 h at 121 °C) or by en- zymatic cleavage with acylase (from Aspergillus species, Sigma A- 2156, incubation 36 h at 37 °C). Purification of the residue was performed by HPLC (5 gm) with formic acid (0.02 mol, pH 3.2, flow rate 1.0 ml/min, Rt = 7.64 rain).

In order to synthesize pyridosine (2_), allomaltol (1_0) [14] (3.8 g, 0.03 mol) and N'-acetyllysine (3.8 g, 0.02 mol) were heated under pressure in 2 N-sodium hydroxide for 24 h at 121 °C. Purification was performed on a silica gel column starting with ethyl acetate/ methanol (1 + 1). In a fraction eluted with ethyl acetate/methanol (I + 7), acetylpyridosine was detected (TLC control, spray reagent iron-III-chloride). After acid hydrolysis and HPLC treatment (as described for maltosine) a compound was obtained with spectral data identical to those of pyridosine (_2) [15].

A 150 gmol sample of maltosine and 150 gmol of pyridosine were dissolved in 100 gl of citrate buffer, pH 2.2; retention time of both substances was 49.0 _+ 2 rain (histidine 52.9 min, lysine 54.2 rain, and phenylalanine 40.5 min).

Preparation of pyridone l l a/ b

Allomaltol (1__0) (2.5 g, 0.02 mol) and propylamine (2.4 g, 0.04 mo o were heated in water (50 ml) for 10 h under reflux. The reaction mixture was extracted with methylene chloride for 8 h. The organic layer was dried over sodium sulphate, concentrated under reduced pressure and the residue distilled at 0.1 Torr and up to 180 °C increasing temperature. The sublimate (1.7 g, 50% yield) was recrystallized from ethyl acetate, l l___g.a: colourless, crysalline substance with mp 198 °C. - 1H-NMR (CDC13): 6 0.99 (t, 3H), 1.78 (m, 2H), 2.37 (s, 3H), 3.79 (t, 2H), 6.31 (s, 1H), 7.16 (s, 1 H ) . - I R (KBr): 1630, 1570, 1520, 1340, 1240 cm -I . - M S : m/z 167 (87%, M.+), 139 (26), 138 (35), 125 (83), 111 (26), 110 (26), 97 (57), 96 (39), 58 (61), 43 (100).

A 0.17 g of sample 1 l~a was dissolved in 2 N-sodium hydroxide (2 ml). After stirring and cooling 0.4 ml of dimethylsulphate was added dropwise and after 1 h at room temperature the solution was heated for 30 rain in boiling water. 1 l__.b_b was extracted with methylene chloride and after removal of the organic solvent, further purification of 111) was performed by TLC (2 ram) with ethyl acetate/methanol (1 + 1). 1 l___.)b was eluated from a band with a R r value of 0.4. 1 l_._.)b: 1H-NMR (CDC13): 6 1.00 (t, 3H), 1.78 (m, 2H), 2.39 (s, 3H), 3.80 (t, 2H), 3.91 (s, 3H), 6.35 (s, IH), 7.12 (s, 1H). - MS: m/z 181 (100%, M.+), 180 (95), 151 (70), 150 (45), 149 (35), 139 (99), 110 (45), 109 (65), 59 (40), 43 (65), 41 (60). The retention time, Rt, with GC system 1 was 11.64 min and 14.61 min with GC system 2.

Preparation of pyridone 12a/b

Isomaltol (2.5 g, 0.02 mol) and propylamine (2.4 g, 0.04 moo were heated in water (50 ml) for 6 h under reflux. Further purification was performed as described for pyridone 11 a/b (1.9 g, 57%). 12a: colourless, crystalline substance with mp 162 °C. 1H-NMR (CDC13): 6 0.98 (t, 3H), 1.78 (m, 2H), 2.39 (s, 3H), 3.90 (t, 2H), 6.39 (d, 1H), 7.23 (d, 1H). - MS: m/z 167 (90%, M+), 152 (79), 125 (90), 97 (47), 96 (68), 58 (68), 43 (100), 41 (68). - IR (KBr): 1630, 1570, 1520, 1340, 1240 cm- 1.

12____aa was methylated as described for l la . 12b: 1H-NMR (CDCla): (5 1.00 (t, 3H), 1.78 (m, 2H), 2.39 (s, 3H), 3.83 (t, 2H), 3.93 (s, 3H), 6.40 (d, 1H), 7.23 (d, IH). - MS: m/z 181 (100%, M+), 180 (44), 166 (69), 149 (56), 139 (88), 138 (81), 136 (56), 124 (44), 121 (75), 111 (44), 110 (94), 96 (44), 43 (94), 41 (75). The retention time with GC system 1 was 3.31 min and 15.04 min with GC system 2.

Isolation of lla and 12____a from a maltose propylamine reaction mixture

Maltose (14.4 g, 0.04 mol) and propylammonium chloride (3.8 g, 0.04 mol) were heated in water (100 ml, pH 7, phosphate buffer) under reflux for 10 h. The reaction mixture was extracted with methylene chloride. The organic solution was concentrated and the residue separated by TLC (2 mm) with ethyl acetate/methanol (1 + 1). A band with an Rf value of 0.6 was eluted with methanol. After methylation of the isolated product, the R t value obtained with GC system 1 was 11.74 rain and 14.65 rain with GC system 2. When compound 11 b was added 5% of 1 Ib was detected (Peaks with Rt = 13.34 min and 15.09 rain, respectively). In the 1H-NMR spectrum of the isolated product only doublets at 6 = 6.40 and 7.24 could be seen.

Formation of 14 from 6

Galactosyl isomaltol [13] (10.8 g, 0.03 mol) and propylammonium acetate (3.6 g, 0.03 mol) were heated in water (40 ml, pH 8, phosphate buffer) and ethanol (30 ml) for 5 h under reflux. Ethanol was removed under diminished pressure and the aqueous solution was extracted with methylene chloride for 10 h in order to separate pyridone 12a. The aqueous layer was concentrated and methanol was added to the residue. Undissolved phosphate buffer salts were fil- tered and the residue was fractionated on a silica gel column with ehtyl acetate and increasing amounts of methanol. With ethyl acetate/methanol (1 + 9) a fraction was obtained containing compound 14. For further purification the syrupy residue was separated by TLC ~.5 mm) using ethyl acetate/methanol (2+ 3). From a band with an R t 0.35 compound 14was eluted with methanol (0.4 g, 4%). 14: colourless, crystalline substance with decomp. 130 °C. 1H-NMR (D20 , 270 MHz): ~ 0.87 (t, 3H), 1.75 (m, 2H), 2.40 (s, 3H), 3.6M.0 (m, 6H), 4.18 (t, 2H), 5.39 (d, 1H), 7.09 (d, 1H), 7.58 (d, 1H) . - IR (KBr): 1600, 1570, 1520, 1330, 1280, 1140, 1080, 1060 cm -1. - M S (FAB): 336 (M+Li) and 328 (M-H). - UV (CH3OH). 2max = 223 nm (loge = 4.39), 267 nm (1oge = 3.6) and 319 nm (loge = 3.94). - Elemental analysis calculated for C, H, and N (monohydrate) was C,d 51.86%; H, 7.25%; N, 4.03%. The value actually found was: C, 51.72%; H, 7.27%; N, 3.98%.

Isolation of 13 from the reaction mixture of l-piperidino maltulose and propylamine

Piperidino maltulose [13] (26g, 0.05 mol) and propylammonaum chloride (19 g, 0.2 mol) were heated in water (120 ml, pH 7.3, phosphate buffer) for 5 h under reflux. Further isolation of 13 was performed as described for 14. Purification of 13 was achieved with HPLC (7 gin, water/methanol (9 + 1) flow rate 1 ml/min, detection at 320 nm). 13 was eluted at an Rt value of 19.96 rain. 13: 1H-NMR (D20, 270 MHz): 6 0.87 (t, 3H), 1.75 (m, 2H), 2.40 (s, 3H), 3.4M.0 (m, 6H), 4.15 (t, 2H), 6.18 (d, 1H), 7.12 (d, 1H), 7.58 (d, 1H).

Results and discussion

We were able to isolate large amounts of the pyridone derivative _8 by the conversion of isomaltol (7) with N%acetyl lysine. The deacetylated product _9 is obtained by acid hydrolysis; analogous to furosine, we have suggested the name maltosine for this compound. By means of the reference substance, it was possible to demonstrate that 8 is also formed by con- versions with disaccharides.

209

0 0 maltose N'--acety!-~, j~OH HCI ~JJ~OH

lactose tysine CH 3 CH 3 I

OH (CH2)4 (CH2)~ ' CH-COOH CH-COOH

COCH~ l I 7 HNCOCH3 NHz =

8 9 Fig. 4 = =

0 0 +oH__ H3C H~C

C3H7 I0 = II a : R = H

=

b : R = CH~ Fig .5

If maltose is heated with N%acetyl lysine in buf- fered, approximately neutral aqueous solution, the formation of 8_ can be readily demonstrated by TLC ow- ing to a sensitive iron-III-chloride reaction. The isolation of 8 from the complex reaction mixture proved to be difficult, since 8 is not readily extractable and cannot be distilled without decomposition. The separation and purification can be carried out inter alia with silica gel and reversed-phase low pressure column chromatography. In this procedure, 8 was obtained with a yield of about 0.1%. 8 is in fact formed in higher yields, but the separation process is associated with high losses. Compound 8 and maltosine (9), respectively, are thus true products of the Maillard reaction, whereas pyridosine (2) is only formed under the conditions of acid hydrolysis.

In the amino acid analysis, it is shown that pyridosine ~) and maltosine (9) cannot be separated on conventional ion exchangers. Therefore, new criteria for the detection of lysine damage must be proposed. In earlier investigations it was noticed that pyridosine and furosine do not occur in a constant quantitative ratio [16]. One reason for this might be that maltosine was not separated so that a higher content of pyridosine was simulated.

In connection with the investigations on the formation ofmaltosine (9), the question arises as to whether pyridosine (2) is indeed only formed in the acid hydrolysis from the Amadori compound, so that it is an "artificial product" in terms of food chemistry, or whether pyridosine as well as maltosine can be formed under the conditions of the Maillard reaction. We have attempted to clarify this in further investigations. Since 2 and 9 can only be separated chromatographi- cally with great difficulty, according to our results so far, the analogous N-propyl derivatives l la and 12a were used for these experiments. 1 l__aa is well accessible in preparative terms via allomaltol (10) and 12a by conversion ofisomaltol (7) with propylamine. 11 a and 12a can be readily separated from sugar reaction mixtures as lipophilic compounds and can be purified by silica gel chromatography. However, chromato- graphic separation of 11 a and 12a proved to be diffi-

cult. Since the protons of the pyridone rings of 1 l____~a and 12a differ markedly in the NMR spectra, an admix- ture of the other compound in one of the components can be detected down to about 5%. By methylation, the O-methyl derivatives l lb and 12b are obtained from 11 a and 12a. These compounds can be separated on capillary columns by GC. However, the corresponding peaks are so close together that an admix- ture of one of the components in the other component can also be detected down to about 5% using this method.

We have heated maltose and lactose with propylamine in approximately neutral aqueous solution and separated 12a from the extract of the lipophilic products by silica gel chromatography. A contamination with 1 la/b could not be demonstrated either by N MR analysis or by GC of the O-methyl derivative 12b. It is thus shown that pyridone 12a is formed either exclu- sively or predominantly, within the specified limit of detection under the condition of the Maillard reaction. If 1 la is formed at all, it is in negligible amounts as compared with those of 12__a. tt can probably be as- sumed that lysine side-chains in proteins react as well as propylamine.

After conversion of galactosyl isomaltol (6) with propylamine, in addition to 12a we also isolated a very polar, crystalline compound which can be regarded as pyridinium betaine 14 on the basis of the spectra. The signals of the two protons on the pyridinium ring (doublets at 7.08 and 7.57 ppm) are especially charac- teristic. These differ markedly from corresponding signals of the O-methyl derivative 12b (doublets at 6.40 and 7.23 ppm). Under the action of acids, pyridone 12a is formed from 14 under cleavage of the galactose residue.

7 ~

Fig. 6

0

fl]2 °R CH 3

I C3H7

12 a : R = H =

b: R= CH 3

210

ma[tose OH lactose HO~-~-o~

NO H~H~Oo

?HrNHC3H7 ~ +1 ~ O - C=O ~ CH3 I 13 C3H 7 HO-CH I

HC-OR I

HC-OH H2C-OH HO /'1

R = ~-glu,13-ga[ HO~__~ OOH 1 . o ~o- 6__ CH~

1=__ ~ C3H7 Fig. 7

12....q

Even after heating 1-piperidinomaltulose and propylamine in approximately neutral aqueous solution, the glycosylated pyridinium betaine 1_3 can be isolated from the reaction mixture in addition to 12a. These compounds 1__3 and 14 are interesting intermediates in the formation of 12a. We are at present investigating further reactions of the pyridinium betaines 13 and 14.

References

1. Paulsen H, Pflughaupt KW (1980) In: Pigman W, Horton D (eds) The carbohydrates: chemistry and biochemistry, vol 1B, 2nd edn. Academic Press, New York, p 881

2. Erbersdobler H, Zucker H (1966) Milchwissenschaft 21:564 Heyns K, Heukeshoven J, Brose K-H (1968) Angew Chem 80:627

3. Finot PA, Brieout J, Viani R, Mauron J (1968) Experienta 24:1097

4. Finot PA, Mauron J (1972) Helv Chem Acta 55:1153 5. Finot PA, Magnenat E (1981) Prog Food Nutr Sci 5:193 6. Severin Th, Loidl A (1976) Z Lebensm Unters Forsch 161:119 7. Kontoghiorghes GJ, Evans RW (1985) FEBS Lett 189:141 8. Nelson WO, Rettig SJ, Orvig C (1987) J AM Chem Soc

109:4121 9. Keeler RF (1975) Lloydia 78:74

10. Erdmann E, Schfifer C (1910) Chem Ber 43:2398 11. Fisher BE, Hodge JE (1964) J Org Chem 29:776 12. Neuberger A, Sanger F (1943) Biochem J 37:515

Zahn H, Huber H, Ditscher W, Wegerle D, Meienhofer J (1956) Chem Ber 89:407 Hardy PM, Nicholls AC, Rydon HN (1976) J Chem Sock Perkin Trans 1:958

13. Hodge JE, Nelson EC (1961) Cereal Chem 38:207 14. Yabuta T (1924) J Chem Soc 575

Brown MG (1956) J Chem Soc 2558 15. Finot PA, Viani R, Bricout J, Mauron J (1969) Experientia

25:134 16. Steinig J, Monta A (1982) Z Lebensm Unters Forsch 174:453

Received August 9, 1988

211

formation of maltosine, a product of the maillard reaction with a pyridone structure

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