part 1
Maillard Reaction
The initial conditions: what we need to get the reaction started
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Author: A. Fabio
This article contains:
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What is the Maillard reaction?
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What are the 3 stages of the Maillard reaction?
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What are the initial conditions to get the reaction started?
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What are reducing sugars?
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What are amino acids?
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What is the antioxidant activity of Maillard reaction products?
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What is pH and how it's involved in Maillard reaction?
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Why is water contained in dough important for Maillard reaction?
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What is the ideal temperature for Maillard reaction?
Introduction
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What is the Maillard reaction?
Maillard reaction is the key phase of baking our pizzas. And it's not exclusive to pizza.
It's often mentioned in tv cooking shows as the reason for a sequence of desired phenomena when cooking and baking. These desired phenomena present as:
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The inclination of some food to brown (in various shades) during cooking (non-enzymatic browning)
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The release of a variety of odours, fragrances and tastes, typical of cooking food (the smell of roasted beef, grilled lamb, freshly baked bread, toasted coffee beans)
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The formation of a superficial crispy crust that is rich in flavors (the crust on the meat cooked on the barbeque, the bread crust or the crispiness of fries).
The desired effects (we will return to the undesired effects later) are the outcome of a chaotic sequence of chemical reactions called 'Maillard Reaction'.
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What are the 3 stages of Maillard reaction?
Maillard reaction can be divided into three stages:
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The reaction starts: 2 agents (amino acids + reducing sugars) that present on the surface of food react with the temperature rising generating a new product called Amadori product. No colour change or odours are generated in this stage.
Subproducts characteristics: emulsifying, foaming, solubility, gelling, heat stability. -
Fission, dehydration, and a reaction called Strecker degradation, generate intermediate products. Odours and aromas are some of these products.
Subproducts characteristics: good smells of food, light browning, antimicrobial, antioxidant, antimutagenic, texture. -
The condensation of the intermediate products generate melanoidins (brown pigments). Undesired products can be generated in this phase.
Products characteristics: browning, potentially mutagenic.​
In short,
the products of the Maillard reaction responsible for the good smell of food appear in the second stage of the reaction and the products responsible for brown colour, called melanoidins appear only in the latter stage of the reaction.
This scheme shows all the complexity of the phases of Maillard reaction. This article goes relatively deep into Maillard reaction. The first part of the present article will analyze the starting condition of the reaction that we see on the top left of the scheme: the reaction between a protein and a reducing sugar.
The purpose of this article is to explain the chemistry behind the reaction in a simple way. The chemical insight can be skipped : essential information for understanding the reaction is highlighted in red.
We cannot deny that the Maillard reaction is extremely complex; a multitude of variables can influence and change the final effects:
the high temperatures that speed up the processes, make them more complicated. This means that some stages of the reaction are still unclear today. For clarity, the purpose of this article is not only to explain the key passages of this myriad of reactions, but also to give some information on how to control the reaction and accomplish the desired effects listed above.
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The initial conditions
(reducing sugars, amino acids, pH, water activity and temperature)
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What are the initial conditions to get the reaction started?
The Maillard reaction starts when reducing sugars and amino acids react in presence of a little water, high temperature and alkaline pH.
​This sentence describes the genesis of the whole process and needs to be analyzed thoroughly before we go any further.
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The reducing sugars
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What are the reducing sugars?
Reducing sugars are any sugar (monosaccharides and some disaccharides) containing a terminal carbonyl group >C=O. This group is very reactive and undergo oxidation reactions (take electrons), which make it ideal for a reaction with the amino group -NH2 (group able to give electrons) of amino acids -Maillard reaction-.
Let's start from the basics:
Sugars, reducing sugars and the redox reactions
Sugar is a generic name for sweet-tasting soluble carbohydrates having the generic formula Cn(H2O)n, where n ≥ 3. This means that there is a water molecule for each carbon C atom. The water molecule is not attached to the carbon C as H2O but as H hydrogen and OH divided: H-C-OH
Glucose C6H12O6 is a simple sugar (monosaccharide). You can observe its linear form, the chain of
H-C-OH groups and the carbonyl group in red, typical of reducing sugars. Other reducing sugars are:
fructose and galactose C6H12O6 (same formula of glucose but different disposition);
glyceraldehyde C3H6O3; many disaccharides (2 monosaccharides bound together) in some forms such as lactose and maltose can be reducing sugars too.
Monosaccharides are the most simple sugars and they are basically chains of H-C-OH groups bounded to each other, with the addition of another group >C=O called carbonyl group. Monosaccharides have minimum 3 carbon C atoms and maximum 9.
You might have noticed that C and O in the carbonyl group share a double bound =.
Chemical bonds
Molecules are groups of atoms held together by attractive forces known as chemical bounds. A single bond happens when 2 electrons belonging to 2 different atoms create a shared orbital that connects the two atoms. A double bond involves 4 electrons and 2 shared orbitals (2 electrons each orbital). Let's keep in mind that electrons have negative charges and protons, located in the nucleus, have positive charges.
Orbitals are area around the nucleus (nucleus is made of protons and neutrons) of an atom where the electrons are likely to be found. The hydrogen has one spheric orbital. Three of the carbon's electrons tend to occupy three hourglass shaped orbitals which are very reactive and can make bonds with various elements.
Bonds can be joined orbitals with shared electrons. Each orbital can host a maximum of 2 electrons. When orbitals have 2 electrons, they are stable and not reactive. Orbitals with one electron are those involved in bonds. In this diagram the hydrocarbon ethane C2H6 is represented. The bound between 2 carbons is highlighted in purple.
The carbonyl group's bonds and polarity
The carbonyl group has a double bond between carbon C and oxygen O. The electron configuration of this group is polarized with a positive pole near the carbon C and a negative pole near the oxygen O. The positive pole on the carbon side attracts chemical species with a negative pole (like the amino group of amino acids) taking their electrons. This type of reaction in chemistry is called reduction, That's the reason why sugars that have the carbonyl group are called reducing sugars.
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Carbon C and oxygen O atom's attractive forces make a double bond and create a carbonyl group C=O. Orbitals can host up to 2 electrons each. Orbitals with 1 single electron, like those shown in the diagram, are not stable but are very reactive. They tend to stabilize themselves ,sharing the lone electron, to make a pair in a joint orbital.
In the carbonyl case the vertical orbitals join together creating a new orbital with 2 electrons shared. That's the first bond of the carbonyl group called σ (sigma) bond.
The second bond, highlighted in yellow, is made by the 2 horizontal orbitals joint together and sharing 2 electrons. This bond is called π (pi). The carbon atom has still the capacity to make 2 more bonds. In sugars, these 2 bonds will be connected to another carbon C and one hydrogen H.
In reducing sugars like glucose, as shown in the diagram, the carbonyl group is connected to the rest of the carbon chain either at one end of it (aldehydes) or in the middle of the chain (ketones).
The presence of 2 orbitals with unpaired electrons on the oxygen side makes a negative pole and consequentially a positive pole on the carbon side. That's why the carbonyl carbon attracts negative species like the -NH2 group of amino acids for the Maillard reaction.
Reducing sugars, the list
All monosaccharides are reducing sugars because they all have a carbonyl group or they are able to form it in alkaline environments. Monosaccharides are galactose, glucose, glyceraldehyde, fructose, ribose, and xylose. Many disaccharides, like cellobiose, lactose, and maltose, also have a reducing form.
Pentose sugars (5 carbon atoms) are more reactive than hexoses (6 carbon atoms) in Maillard reaction. Disaccharides react the least.
Pentose sugars are: ribose, xylose, arabinose.
Hexose sugars are: galactose, glucose, glyceraldehyde, fructose.
In short,
The efficiency of reducing sugars undergoing browning reactions follows this order:
Ribose > Xylose > Arabinose > Fructose = Glucose > Sucrose > Maltose (1)
Sucrose, the common table sugar, is not a reducing sugar. It's a disaccharide made of glucose and fructose. It can take part in Maillard reaction only after the bond between glucose and fructose is broken down by the enzyme amylase, naturally present into flours.
The lack of reducing sugars
A lack of reducing sugars can be the reason why pizzas turn out too pale:
Yeast and lacto-bacteria (sourdough) use simple sugars present in the flour to activate their metabolism. If the temperature is too high for the amount of yeast used in the dough or the dough is left proofing for too long, the yeast might eat most of the simple sugars present. That can cause a lack of reducing sugars needed to start the Maillard reaction. Balancing the amount of yeast used and the proofing time is again essential; but also using a flour with the addition of the enzyme amylase can help to decompose the starch into simple sugars.
The amino acids
Amino acids are the other side of the beginning of the Maillard reaction.
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What are the amino acids ?
Amino acids are a group of 20 molecules that combined together in chains make proteins. All amino acids are characterized by two reactive groups: the amine group -NH2 and the acid carboxyl group -COOH.
The group that we are going to keep an eye on in the Maillard reaction is the amine group.
The amine groups
The amine groups are functional groups that contain a basic nitrogen N atom with a lone pair (2 free orbitals with one electron each not bonded to any element). Due to the lone pair, amino groups easily react to species with a positive pole like the carbonyl group.
Amine groups in amino acids are called primary amine when the nitrogen atom is bonded to two hydrogen atoms -NH2.
Secondary amines with only one hydrogen or tertiary amines without any hydrogen are less reactive in Maillard reaction than primary amines, also called amino group -NH2.
Two amino acids: glycine (above) and lysine. The acid carboxyl group is highlighted in red. The amine group that is functional to the Maillard reaction is highlighted in green. Lysine is the most reactive amino acid for Maillard reaction because it has 2 amino groups -NH2 well exposed.
The amino acid proline has a secondary amine (nitrogen is bounded to only one hydrogen H atom). Primary amines -NH2 are more reactive because they are more physically exposed. Proline will take part in the Maillard reaction as well but at a slower rate.
All free amino acids can take part in Maillard reaction. The Amino acids part of proteins can only take participate in Maillard reaction if the protein has a primary (linear) structure as that's the only structure that has the amino groups well exposed. If proteins have a secondary or a tertiary or a quaternary structure, the amines are not exposed but they are hidden in the spirals of the protein's structure. In general free amino acids are far more reactive than proteins.
Primary, secondary and tertiary structure of proteins are represented in the diagram. The primary structure is a linear chain of amino acids with the functional groups -NH2 well exposed on the sides. The secondary structure has a 3D structure with inner interactions between the amino acids (hydrogen bonds) that give the structure the shape of an helix or a pleated sheet. The amines are not exposed anymore. the tertiary structure is the overall three-dimensional shape that is formed by the interactions of the side chains of the various amino acids and made of linear, helix and pleated sheet sequence of amino acids.
The amino group -NH2 is the most reactive of the amines in amino acids because it's the most exposed. The lone pair of electrons on the left is shown in the picture. Those make the group a good nucleophile (able to donate electrons) and attack the carbonyl carbon.
The reaction between a reducing sugar and an amino acid happens between the two functional groups, carbonyl group >C=O and the amino group -NH2 . The positive pole around the carbonyl carbon C atom attracts the negative pole of the amino nitrogen N and the 107° angle C-C-O, highlighted in orange in the picture (Bürgi–Dunitz angle) is a perfect fit for the nitrogen N. The weak π bond (the second bond, highlighted in yellow) between the carbon C and the oxygen O of the carbonyl group will break because the attraction force of lone orbitals of the nitrogen N is higher.
The reducing sugar represented is glucose and the amino acid is glycine.
The reaction has started and the first compound has been finally formed: a glycosylammine. The π bond has been broken and now the oxygen has an extra spare orbital with a single electron, which makes that pole even more negative. Carbon and nitrogen bonds together the reducing sugar and the amino acid.
Amino acids and the antioxidant activity of Maillard reaction
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What is the antioxidant activity of Maillard reaction products?
The products of Maillard reaction can be an important source of antioxidants. It's been proven that an higher amount of amino acids makes a darker crust (more melanoidins are formed) and a higher antioxidant activity.
Bread or pizza dough with glycine and/or lysine and/or alanine added has more melanoidins and higher radical scavenging activity against free radicals after cooking.
It seems like the antioxidant activity in white bread is correlated with the browning level. (2)
The UV absorbance of the melanoidins generated from Maillard reaction (the browning levels) with different amounts (0.1g, 0.3g, 0.6g) of 6 amino acids. Glycine makes bread with a darker crust.
The antioxidant activity of products generated from Maillard reaction (anti-free radical activity) with different amounts (0.1g, 0.3g, 0.6g) of 6 amino acids. Glycine, lysine and alanine makes bread crust with a higher antioxidant activity.
White bread flours are generally poor in lysine. Lysine, glycine and alanine able to generate good antioxidant products in the Maillard reaction in wheat flour are found in glutenin proteins. Strong flours are generally richer in glutenin. Glutenin has a tertiary structure which makes not very reactive. It's very important to consider the maturation phase of the dough to have access to free amino acids. During the maturation, enzyme protease attack and partially decompose proteins into simple amino acids that can take part into Maillard reaction.
Amino acids and accessibility in fermented bran and germ
The addition of fermented bran and/or germ is the trademark of the pizza dough of The Secret Pizza Society.
Both fermented bran and fermented germ add a considerable amount of free amino acids to the dough.
The total amount of free amino acids increases by 43% from non fermented bran to fermented bran. Among all the amino acids that significantly increase in bran fermented with lactobacillus strains, there are glycine, lysine and alanine. (3)
We have similar outcome in terms of amino acids increase and consequently Maillard reaction rate with the addition of fermented wheat germ. (4)
In short, the addition of fermented bran and/or germ helps Maillard reaction.
The effect of different addition levels of fermented wheat germ on the bread color (2%, 4%, and 6% wheat flour was replaced by fermented wheat germ flour)
pH in Maillard reaction
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What is pH and how it's involved in Maillard reaction?
Maillard reactions occur under alkaline condition. The ideal pH for a good reaction is around 6-8.
What is pH?
PH is a scale used to specify the acidity or basicity of an aqueous solution. The scale goes from 0 to 14 where 0 indicates the most acidic solution like battery acid, 7 neutral solution like pure water, and 14 the most basic like liquid drain cleaner.
The pH scale measures whether there is more hydronium H+ or hydroxide OH- in a solution. These two ions come obviously from water H2O that can lose a proton H+ (an hydrogen atom without its own electron) and split from the negative ion OH-. A solution that has more ions H+ than OH- is called acidic and vice-versa, a solution that has more more ions OH- is called basic. Most foods are acidic (Ph < 7).
The role of pH in Maillard reaction
Acidic pH inhibits Maillard reaction because the amino group -NH2 can react with the hydronium H+ (of which acidic solutions are rich) becoming the ammonia group -NH3 instead of reacting with the carbonyl group of the reducing sugar. Therefore, the Maillard reactions doesn't happen for that amino acid.
The reactivity of the amino acid is influenced by the pH. Put simply: At low pH the amino group is protonated (has another hydrogen H atom, attached to one of the 2 reactive orbitals) and -NH2 becomes -NH3. -NH3 has a positive pole and is not attracted to the positive pole around the carbonyl carbon. That's why the reaction doesn't happen. The reason why the amino group -NH2 gets protonated in acidic environment is that acidic pH means a higher presence of ions H+ in the solution as shown in the picture. The more acidic the solution is, the more H+ ions are in the solution. It is more likely that one of them attached to the amine group: NH2 + H+ -> NH3
An effective way to inhibit Maillard reaction is to acidify the product: marinate in wine, vinegar, lemon.
Sourdough or preferments like biga or poolish, make the dough more acid, therefore bread or pizza can be less brown.
Adding cooking ammonia or baking powder/bicarbonate soda to products makes them more alkaline and that helps Maillard reaction. A sourdough starter that is too acidic or dough that is too old can make pizzas or breads too pale.
Water activity
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Why is water contained in our doughs important for Maillard reaction?
Water activity is the presence of free water in the dough, as free molecules of H2O not bounded to any other compound. The presence of water activity works as the field where the amino acids and the reducing sugars travel. If that’s too high our agents are too dissolved and it’s too difficult for them to meet. The water activity scale goes from 0 (total dryness) to 1 (just water) and the ideal value of water activity for Maillard reaction is 0.6.
The temperature
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What is the ideal temperature for Maillard reaction?
The ideal temperature for Maillard reaction is above 140 C. At that temperature the molecules have enough energy to move fast and collide into each other and the proteins tend to unfold their structure and expose the amino groups for the reaction.
At lower temperatures, the reaction might still happen if other conditions are ideal enough, but much slower or with less noticeable effects.
The tanning products for example are a mixture of proteins and reducing sugar that don’t need high temperature to brown.
Roughly every 10 degree Celsius increase in cooking temperature (after the Maillard starting temperature is reached, which is varies from food to food, is 140 C for bread and pizza) doubles the amount of the Maillard reaction products (5). That leads us to be careful, if it gets too hot, food is heading towards the irreversible pyrolysis reaction (burning).
Conclusion of the first part
To increase the reaction effects:
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add reducing sugars add reducing sugars to the dough like glucose, maltose; use flours rich in starch, or with amylase enzyme added; do not use excessive yeast or sourdough.
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add amino acids use flours rich in proteins (glutenin in particular), strong flour and give the dough a proper maturation time. Add a ferment made of fermented bran or germ to the dough.
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remove water. Reducing the hydration of the dough makes reagents meet faster.
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increase temperature the temperature plays a key role in Maillard reaction, higher the temperature faster the reaction happens
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pH control of pH is another way to control the reaction. Adding alkaline elements to the dough such as baking powder, bicarbonate soda (not recommended in pizza dough) or being careful with the acidity level of dough (old dough tends to acidify) is a powerful way to control the reaction.
In the second part of the article, further stages of the reaction will be analyzed, included the formation of undesired products. An insight into the odour of the freshly baked bread and the formation of a perfect crust will be explored.
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References
1 - Delphine Laroque, Claude Inisan, Céline Berger, Éric Vouland, Laurent Dufossé, Fabienne Guérard (2008)
Kinetic study on the Maillard reaction. Consideration of sugar reactivity
2 - Thinzar Aung, Seung-Soo Park and Mi, Jeong Kim - Changwon National University, Republic of Korea -
Influence of Lactobacillus (LAB) Fermentation on the Enhancement of Branched Chain Amino Acids and Antioxidant Properties in Bran among Wheat By-Products
3 - Yanting Shen, Lauren Tebben, Gengjun Chen & Yonghui Li (2018) International Journal of Food Science & Technology -
Effect of amino acids on Maillard reaction product formation and total antioxidant capacity in white pan bread
4 - Yansheng Zhao, Jiayan Zhang, Yixing Wei, Lianzhong Ai, Dong Ying, and Xiang Xiao -
Improvement of Bread Quality by Adding Wheat Germ Fermented with Lactobacillus plantarum
5 - Brady, J. (2013) Ithaca, New York: Cornell University.
Introductory Food Chemistry.
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