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Analysis of Maillard reaction products


Ansynth Service B.V. is highly specialized in analysis of furosine, analysis of carboxymethyllysine (CML) and analysis of hydromethylfurfural (HMF) as indicators of Maillard reactions in a wide variety of products. This document is a very brief guideline to clarify the complex world of Maillard reactions.

Lysine is one of the most essential amino acids and fairly limited in a lot of food and feed stuff and the first limited amino acid in diets for production animals. Lysine possesses a reactive amino group in the end of its side chain (ε-amino group) which can react with other compounds to form nutritionally unavailable products, most common: Maillard products when reducing sugars are involved. Other amino acids are also thought to undergo Maillard type reactions, for example, proline, tryptophan and arginine but these reactions are less well studied. Maillard reactions products are named after its finder: L.C. Maillard who reported in 1912 about color formation in aqueous solution with sugar and amino acids, also known as non-enzymatic browning, which should not be interchanged with caramelisation, another type of non-enymatic browning. Since further acknowledgement of Maillards findings in the fifties, the importance of Maillard chemistry has been widely shown in food science, nutrition and medicine.

In human nutrition, most protein sources have been processed in a factory and are probably heat treated, which facilitates the formation of Maillard reactions. Although these reactions occur at any temperature, the rate is significantly accelerated at higher temperatures.

Lysine that reacts with other compounds is not available anymore for digestion in the organism. Basically the occurrence of Maillard reaction leads to:
  1. Loss of nutritional quality, due to the availability of lysine.
  2. Loss of safety. Toxic and physiological effects have been reported about the presence of Maillard reaction products. But also compounds with anti oxidative and anti mutagenic effects been reported in milk and milk products.

The scheme of Maillard reaction is quite complex and consists in general of 3 stages:
  • Early stage, formation of an (unstable) Amadori compound: fructosyl-lysine (from glucose) or lactulosyl-lysine (from lactose) or maltulosyl-lysine (from maltose).
  • Advanced stage, breakdown of an Amadori compound, resulting in a variety of products which formation is pH dependent. One significant compound to be formed at lower pH is hydroxymethylfurfural (HMF). At higher pH it’s thought that the “Strecker degradation” will occur where di-carbonyl compounds react with amino acids to yield aldehydes and carbondioxide eventually. In addition, it’s thought that 1,2-dicarbonyls targets the guanidine side chain of arginine, resulting in numerous possible compounds.
  • Final stage, formation of melanoidins from reaction products and amino compounds resulting in a typical brown colour and flavor enhancement.


Analysis of Furosine

A clear marker of early Maillard reactions is the compound furosine: ε-N-(furoylmethyl L-lysine, formed by acid hydrolysis of an Amadori product and therefore serves as a suitable marker of process and food quality. However during amino acid analysis, when acid hydrolysis is applied to determine the available lysine content, the Amadori product is partially reverted to lysine, meaning: the value found after acid hydrolysis as performed in a regular analysis does not necessarily reflect the total amount of available lysine. The meaning of the terms “available lysine”, “total lysine” and “reactive lysine” give often misunderstanding. Reactive lysine refers to lysine residues that hasn’t undergone any Maillard reaction and can therefore be considered as “available lysine”, meaning it can be adsorbed and utilized in protein synthesis. Total lysine refers to reactive lysine + blocked lysine together.

Analysis of Carboxymethyllysine (CML)

Another type of Maillard reaction products is “Advanced Glycation Endproducts” (AGEs) which occur in vivo (human body) where it’s related to pro-oxidative and pro-inflammatory effect but does occur in nutritional products as well. Reaction of the ε-aminogroup of lysine with the oxidation product of glucose, glyoxal or methylglyoxal, leads to formation of N-carboxymethyllysine (CML) or N-carboxyethyllysine (CEL). As a secondary route, CML can be also formed by oxidation of the Amadori product fructosyl-lysine. CML is formed in food through various pathways and can be determined as free form as well as protein-bound form. Next to furosine, CML serves as suitable marker food quality, especially for those products that has been more severely heated or has a higher content of fructose and where determination of furosine solely would result in under-estimation of the heat damage.

Analysis of Hydroxymethylfurfural (HMF) and Furfural compounds

The pathway that leads to formation of HMF consists of 2 possible routes.
  1. Continuation of the Maillard Reaction from an Amadori product, meaning that HMF is a good indicator for Maillard Reactions in advanced stage.
  2. Sugar degradation and caramelization, facilitated by heat treatment. This route especially works with sucrose and fructose and is accelerated under acidic conditions. This type can happen during storage of food products under inappropriate conditions. This second route is independent from the first route.

HMF is definitely a clear marker of more severe heating and is analyzed in numerous products like - - Milk and milk products like milk powders and infant formulas
  • Honey and syrups
  • Tomato paste
  • Bakery products
  • Roasted products like coffee beans

HMF can be measured as “free form” but also as “total” after hydrolysis, better described as “potential” HMF. The short hydrolysis will release protein bound furfural and furfural from Amadori products.

Product storage, shelf life and Maillard reactions


Maillard reactions happen during heating processes such as in a factory but also during long term storage of products. The reaction rate during storage is depending on water content, time and temperature, but the presence of reducing sugars is a requirement. A variety of products is prone to degradation such as milk powders, (enzyme treated or hydrolyzed) cereal products from different origin, animal feed, etc. HMF is known to be present in high levels in roasted food, in dried fruits and high acidic foods like balsamic vinegar. Storing those foods will only lead to higher levels of HMF. Susceptible foods for increasing HMF levels during storage are those that are high in carbohydrates like fruit juice concentrates, honeys and jams and jellies but also increase of HMF levels in infant formulas has been reported.

As regular acid hydrolysis will not be always sufficient to show a decrease in lysine, analysis of Maillard reaction products is a great indicator.

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