Using Proteomics Analysis to Uncover Protein Glycosylation

Our recent work on protein glycosylation has received attention from Following the full text.

Protein recovered from poultry and fish by-products is becoming more important as an ingredient in the food industry. Unfortunately, it is often degraded during the recovery process, reducing quality and usability. However, researchers are developing effective methods to improve recovered protein quality.

One of these techniques, based on the Maillard reaction, modifies proteins by covalently attaching carbohydrates under dry heat conditions. Unfortunately, although the process is effective, it is also slow, difficult to control and can result in formation of known mutagens such as advanced glycation end products (AGEs). These AGEs can develop during storage, even after the initial Maillard reaction has been stopped.

Instead of the dry process to glycate proteins, Hyrnets et al.1 investigated an aqueous state reaction to enzymatically add the amino sugar glucosamine (GlcN) to the protein. The reaction is catalysed by transglutaminase(TGase), an enzyme widely present in a many body tissues. This reaction takes place quickly at lower temperatures and is easier to control, producing a higher quality end product that is more stable during storage.

Picture4First the researchers extracted their protein substrate, natural actomyosin (NAM), from chicken breast muscle before incubating it with glucosamine. They performed the reaction under various temperatures and sugar concentrations to discover optimal conditions for the process. Once processed, the quality of the glycosylated protein end-products was assessed, and the research team used proteomic analysis techniques to determine the success and completeness of the enzymatic reaction.

Firstly, Hyrnets and co-workers evaluated glycoconjugation using matrix-assisted laser absorption/ionization (MALDI) – tandem time-of-flight (TOF/TOF) mass spectrometry to estimate how many GlcN residues had linked covalently with NAM. The optimal reaction environment for maximal binding was 37°C with a 1:1 ratio of sugars to protein; 15% of the peptides available were bound.  The research team also used this assay to examine how TGase glycosylation compared to and competed with glycation by the Maillard reaction. At lower reaction temperatures of 25°C, glycation by the Maillard reaction was reduced in the presence of TGase, resulting in higher levels of the more stable glucosamine-actomyosin bond. This is beneficial during storage as Maillard reaction glycoconjugates are not so stable and continue to form AGEs.

Hyrnets et al. used liquid chromatography-mass spectrometry (LC-MS) to determine the actual site of enzymatic glycosylation on the proteins. Glycosylation catalyzed by TGase occurs at specific sites in the peptide sequence, so by comparing control with glycosylated samples, researchers could accurately define where and how the conjugation was occurring.

The researchers used sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to separate proteins, then digested gel regions of interest with trypsin. Samples were analyzed by nanoflow LC (Thermo Scientific) coupled to an LTQ XL-Orbitrap hybrid MS (Thermo Scientific). Peptide fragments were identified in the resulting MS data using the Proteome Discoverer 1.3 (Thermo Scientific) and the Uniprot protein database (SEQUEST, Thermo Scientific). LC-MS results confirmed that covalent binding of glucosamine with NAM was indeed due to TGase activity.

The researchers also measured quality of end product to show how enzymatic glycosylation performed compared with traditional protein modification. They found that solubility, emulsifying ability, and thermodynamic stability all increased in the glycosylated products, suggesting that the enzymatic glycosylation process could improve the additive ingredient making it more acceptable to the consumer.


1. Hyrnets, Y., M. Ndagijimana and M. Betti 2014.Transglutaminase-catalyzed glycosylation of natural actomyosin (NAM) using glucosamine as amine donor: Functionality and gel microstructure,Food Hydrocolloids 36 (pp. 26-36)”

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Cultured Meat: A possible sustainable source of animal proteins?

My former student, Isha Datar, explains to us (with numbers) why mass production of beef would be unsustainable. The solution would be moving toward the “artificialization” of our food chain by using tissue engineering techniques, like, for instance, cultured meat technology. This technology seems to have the potential to deliver cheap and nutritious meat proteins to a growing population in a sustainable manner.

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La Repubblica delle Idee 2012 – Scrivere il Futuro

Next: il futuro e’ tornato (mio intervento sulla high tech bistecca al minuto 64)

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Creating the perfect partial salt replacement

Mirko Betti shows the traditional kokumi product in his left hand and the cleaner kokumi product he and his team created with a more efficient technology in his right hand.

In the quest to lower sodium consumption in the North American diet, a team of University of Alberta researchers recently received $340,000 to conduct sensory and taste trials of the salt flavour enhancement product it created with a new, cleaner and more efficient technology.

The team took proteins from low value parts of poultry, fish and vegetables and created molecules that have kokumi characteristics. Kokumi was recently identified by the Japanese as the sixth basic taste, an addition to salty, sweet, sour, bitter and umami (or savoury). Translated often as “heartiness” or “mouthfulness,” kokumi describes compounds in foods that don’t have their own flavour. Rather, they enhance the flavour with which they’re combined.

“Hopefully, we’ll be able to significantly reduce the sodium in several food products by replacing it with the kokumi we developed. Because the kokumi amplifies the taste of the salt, it allows foods to have much less salt and be better for you, without sacrificing the flavour. Done right, most consumers wouldn’t know the difference,” says Mirko Betti, who leads the team that also includes Michael Ganzle, Andreas Schieber and Maurice Ndagijimana.

While the flavour enhancer is one product among others that allows food manufacturers to replace salt without sacrificing flavour, kokumi is considered the best because it provides the best punch or first impact of a food, the best mildness and the best long-lasting taste development.

Kokumi is already sold on the market to food manufacturers as a salt enhancer by at least one major international food and chemical company who creates it from soy beans. However, the traditional way in which kokumi is manufactured also leads to the creation of many unhealthy by-products.

What makes Betti’s kokumi unique is the way in which he manufactured it.

The ALES team broke the proteins from the various sources into their component fragments as is usually done. It then selected specific fragments and mixed them with sugars but instead of using the typical heat transfer process to create the kokumi molecules, it used a fermentation process, thereby drastically reducing the unwanted by-products and making the process much more cost-effective.

Plans are now underway to use the funding to conduct sensory and taste trials to fine tune the technology.

The potential for the kokumi market is staggering as consumption of the food enhancer isn’t linked to the ill effects, including heart disease, associated with overconsumption of sodium, which is common in the North American diet.

According to Health Canada, Canadians consume twice the amount of sodium they need every day. While it’s an essential part of a healthy diet, too much sodium can lead to high blood pressure, a major risk factor for stroke, heart disease and kidney disease. Overconsumption of sodium has also been linked to increased risks of osteoporosis, stomach cancer and the severity of asthma.

The funding, which was provided by the Alberta Livestock and Meat Agency and Alberta Innovates – Bio Solutions, gives the team a two-year window in which to conduct the trials and refine its technology to eventually patent and sell it.

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“Kokumi” flavour enhancers from poultry and fish proteins

Dr. Mirko Betti and co-applicants from University of Alberta have received $ 340,000 from Alberta Innovates – Bio Solution to find new technologies to generate kokumi sensation from undervalued poultry and fish proteins.

The term kokumi refers to the Japanese concept relating to the capacity of an ingredient to improve the taste of food. Kokumi is considered the sixth basic taste to which the following three types of flavour sensations are attributed: mouthfulness and continuity (long-lasting taste development), punch (first impact) and mildness. Kokumi molecules have the ability to enhance salt perception and thus allow reduced salt concentrations in foods.

Reduction of dietary sodium is a priority for Health Canada and the Canadian food industry. The average consumption of dietary sodium by Canadians is estimated at 3500 mg/day (Barr 2010). Reducing dietary sodium by 1840 mg/day is recommended to reduce the blood pressure by 5.06/2.70 mmHg in adult hypertensive patients.  Therefore, strategies to replace sodium in processed foods without compromising the taste include the use of salty taste and flavour such as glutamate or kokumi substances.

The main objective of the present project is the development of a new process for the production of new molecules having salty and/or kokumi tastes. Proteins from poultry and fish processing by-products as well as some undervalued vegetable proteins will be used to generate kokumi sensation.

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Food for Thought

Mirko Betti university researcher links philosophy and food
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Can we reduce salt in meat products? Yes we can…with beta-glucans and High Pressure Process Technology

Oats: an important source of beta-glucans

As consumers wish to engage in healthier eating without sacrificing the tastes and textures they are used to, food scientists like ourselves are looking for alternative ingredients and processing methods to create healthier familiar foods.

Sodium has recently become subject to much negative attention due to its association with hypertension and cardiovascular disease, and thus reducing sodium content has become a goal in several processed foods. Reducing sodium content in fast, frozen and snack foods is a relatively simple task. Reducing sodium in processed meats is not as simple.

While salt contributes to flavour, it most importantly contributes to arguably the most important characteristic of processed meats: texture. Meat gelation involves the unwinding of salt-soluble meat proteins and their subsequent aggregation. Salt helps to increase protein solubility. When salt is reduced in processed meats, the elasticity of the meat decreases as well.

High pressure processing is a relatively new technique in the food industry. It is able to inactivate microorganisms and therefore increase shelf life of several different types of foods. In processed meats, it is able to increase water binding and protein-protein interactions, two characteristics important for creating a desirable protein gel.

While high pressure processing can aid in gel formation, it does not suffice on its own. Our study looks at beta-glucan as a partial salt replacement in high-pressure processed chicken. Beta-glucan is a dietary fibre (polysaccharide) naturally derived from several foods; in this case, oats. Fibre has many pronounced health benefits and the FDA recommended daily intake is 3 g. Protein/polysaccharide complexes can increase protein solubility; a necessary step in the formation of a desirable meat gel.

Our studies published in Innovative Food Science and Emerging Technologies and Food Chemistry showed that a chicken meat gel with 1% NaCl and 0.3% beta-glucan has comparable hardness to a gel with 2.5% NaCl when processed at 40 C and 400/600 MPa. This  is promising evidence suggesting that beta-glucan can be used as a partial salt replacement in processed meats while maintaining the mouthfeel of full-salt products.

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Edible Polymer Films from Isolated Muscle Proteins (IMP)

Read the article A New Film Star

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