Chapter 19
Haptenic Sugar Antigens as Immunochemical Markers for the Maillard Reaction of Processed and Stored Milk Products 1
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Downloaded by UNIV OF ROCHESTER on November 15, 2016 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1996-0631.ch019
T. Matsuda and Y. Kato 1
Department of Applied Biological Sciences, School of Agricultural Sciences, Nagoya University, Chikusa-ku, Nagoya 464-01, Japan Department of Clinical Nutrition, Kawasaki University of Medical Welfare, 288 Matsushima, Kurashiki, Okayama 701-01, Japan
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Milk proteins are chemically modified by amino-carbonyl reaction (Maillard reaction) with lactose during processing and storage. We have obtained rabbit antisera and mouse monoclonal antibodies against the Maillard reaction products between lactose and protein amino groups. These antibodies specifically recognize the lactose-lysine reaction products as haptenic sugar antigens, and are useful to detect the Maillard reaction products in processed and stored milk samples. Some market milk samples were boiled for 30 min, while several powdered milk products were kept at 25 °C for a few weeks. The Maillard reaction products in these samples were analyzed by an enzyme immunoassay ELISA. The results indicated that the Maillard products already existed in these market milk and milk products and were further produced by heating of the market milk samples and storage of the powdered milk samples. Proteins are chemically modified with reducing sugars by Maillard reaction between amino- and carbonyl-groups during processing, storage and cooking of various food (7). The Maillard reaction between sugars and proteins as food constituents affect the food quality, e.g., reduction of protein nutritional value, desirable or undesirable browning of food, alteration of functional properties of food proteins. The Maillard reaction of lactose and proteins in milk has long been investigated mainly from a view point of nutritional quality of pasteurized and powdered milk or milk products (2). Availability of an essential amino acid, lysine, is decreased by the reaction with lactose during heating for pasteurization and spray-drying for powdering of milk. Loss of available lysine and production of reaction intermediates are estimated by amino acid analysis of acid hydrolysates of milk samples. Immunochemical methods using specific antibodies are now widely used for analyses to detect a specific component in complex mixtures of food components. The authors have found that novel antigenic structures are produced by Maillard reaction of proteins with lactose (3) and obtained rabbit polyclonal and mouse monoclonal antibodies which specifically recognize lactose-protein Maillard reaction products (4,5). These antibodies not only with lactose-milk protein reaction products but also with lactose-egg protein reaction products, indicating that these antibodies mainly recognize lactose-derived antigenic components in the Maillard
reacted
0097-6156/96/0631-0221$15.00/0 © 1996 American Chemical Society Lee and Kim; Chemical Markers for Processed and Stored Foods ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
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CHEMICAL MARKERS FOR PROCESSED AND STORED FOODS
CH OH 2
Lactulose
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Methyl-p-galactoside Lactitol
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Downloaded by UNIV OF ROCHESTER on November 15, 2016 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1996-0631.ch019
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Galactose Lactose
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Figure 1. Sugars and sugar derivatives used as competitors for the competitive inhibition ELISA.
Figure 2. Inhibition of the antibody binding by Maillard reaction products of lactose with some amino acids. The lactose-amino acid Maillard products were used as competitors against binding of the monoclonal antibody L101 to lactose-protein Maillard products.
Lee and Kim; Chemical Markers for Processed and Stored Foods ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
19. MATSUDA & KATO
Haptenic Sugar Antigens in Milk Products
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reaction products. Such lactose-derived antigens could be an immunochemical marker to evaluate quality of market milk and powdered milk products concerning Maillard reaction caused by processing and/or storage (6). In the present study we investigated the antigen binding specificity of one monoclonal antibody, L101, specific for the lactose-protein Maillard products, and applied the monoclonal antibody to estimate Maillard products in various milk samples.
Downloaded by UNIV OF ROCHESTER on November 15, 2016 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1996-0631.ch019
Epitope structure recognized by the specific antibody The products of Maillard reaction between lactose and protein amino groups are complex mixtures of many unidentified compounds. Among them, a relatively stable and dominant intermediate compound is ε-Ν-deoxylactulosyl-lysine which is the Amadorirearrangementproduct (7). To estimate an epitope structure recognized by the monoclonal antibody L101, competitive inhibitory activity of several sugars (Figure 1) against the antibody binding to the Maillard product were examined by competitive ELISA (5,8). The strongest inhibitor was lactulose, which is a structural analog of the Amadori rearrangement product of the lactose-amino group Maillard reaction. Methyl^-galactoside, lactitol and lactose showed inhibitory activity but their effectiveness was much lower than that of lactulose. Methyl-oc-galactoside, galactose and fucose showed no inhibitory activity under the experimental condition used. Lactulose has been reported to be produced from lactose in large amount during the heating of milk (9), and such lactulose would interfere the antibody binding to the haptenic sugar antigens in milk. However, free lactulose in heated milk samples can be eliminated from the solid-phase immunoassay system such as ELISA, because only protein-bound sugar antigens but not free lactulose can be adsorbed on the surface of the ELISA plate. Therefore, the lactose-protein Maillard products can be analyzed immunochemically without interference of lactulose produced in heated milk products. Contribution of amino acid moiety to the epitope structure was estimated by the competitive ELISA using the Maillard reaction products of lactose with oc-N-acetyl-Llysine, γ-aminobutylic acid, β-alanine and glycine. As shown in Figure 2, the Maillard reaction products of lactose with oc-N-acetyl-lysine, γ-aminobutylic acid, βalanine strongly inhibited the antibody binding, whereas the product of glycine with lactose showed no inhibitory activity. This indicates that not only amino group but also a part of alkyl chain of the lysine residue contribute the epitope structure of the lactose-protein Maillard product. The monoclonal antibody, L101, is also suggested to be raised against the Maillard product of lactose with ε-amino group of lysine residues but not against the product with α-amino group of the N-terminal amino acid residue of proteins. Lactose-derived sugar antigens in commercial milk products Maillardreactionof lactose with milk proteins proceeds during processing such as pasteurization and drying of milk. Apparent content of lactose-derived haptenic sugar antigens in several market milk and powdered skim milk samples was estimated by ELISA using the monoclonal antibody L101. Appropriately diluted or dissolved milk samples were directly used as antigen solution for coating of ELISA plates. Figure 3 shows ELISA values of severalrepresentativemilk samples. The powdered milk samples showed relatively higher ELISA value, indicating their higher content of the lactose-derived haptenic antigen. Among pasteurized market mik samples tested, UHT-milk pasteurized at 140 °C for 3 sec showed stronger positive reaction, and UHT-milk pasteurized at 140 °C for 2 sec and HTST milk pasteurized at 85 °C for 15
Lee and Kim; Chemical Markers for Processed and Stored Foods ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
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CHEMICAL MARKERS FOR PROCESSED AND STORED FOODS
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