Heat Treatment, colour development and Transglutaminase

Heat Treatment, colour development and Transglutaminase

**still working on it**

We are still working on it


The issue of heat treatment relates to colour development, moisture loss, and protein denaturing and gelation.  In this context, we will then consider the optimal temperature and application of TG.


CodeCogsEqn (11) to CodeCogsEqn(8) to NO 

When sodium nitrite is placed in solution in the brine preparation phase, the crystal structure breaks up and the ions separate into Na and CodeCogsEqn (4).  Nitrous acid is formed.    This hydration of nitrous acid is an important time-consuming reaction (Krause, B. L.; 2009: 9).

After the formation of nitrous acid (CodeCogsEqn (11)), the next step “is the generation of either a nitrosating species or the neutral radical, nitric oxide (NO).”  (Sebranek, J. and Fox, J. B. Jn.; 1985:  1170)  A nitrosating species is a molecular entity that is responsible for the process of converting organic compounds into a nitroso (NO) derivatives, i.e. compounds containing the R-NO functionality.  During resting, the most important one is the formation of Nitrosyl Chloride (NOCl).  This is one of the good reasons why leaving out salt from bacon curing is not advisable.

We found that an optimal processing sequence is injecting the meat, tumbling it, resting it for between 12 and 24 hours (depending on the curing room temperature), tumbling it again to pick up brine that leached out during the maturing or colour development stage and then smoking it.  Where meat grids are being used, another option will be to inject, tumble, fill into grids and then resting it, but for this one would require additional trolleys.  (Reaction sequence)


Cold smoking is normally seen as smoking where the core temperature will remain < 25 or 30 deg C.  Different sources, pins the limits at different temperature points.  We use hot smoking where the core temperature riches > 35 deg C but < 45 deg C, which is considered “warm smoking.”  Smoking and thermal treatment are therefore considered jointly.


During reddening, the temperature is increased, extraction flaps in the smokehouse closed, to maintain humidity, and sulfhydryl groups are released which is a reducing substance in meat and important in proper cured colour formation.  Fraczak and Padjdowski (1955) indicated that 80°C is the critical temperature for the decomposition of sulfhydryl groups in meat.” (Cole, 1961)  (Reaction sequence)


During heating and smoking there are several changes in the meat that has a direct effect on the colour development.  The nitrosating species that is more dominant than NOCl is smoking due to the presence of phenolic compounds.  In addition to the heat release of sulfhydral groups, the pH is reduced in the meat.  Randall and Bratzler (1970) noticed an increase in the myofibrillar protein nitrogen fraction, pH and free sulfhydryl groups of pork samples that were only heated, and a decrease of these values in the samples that were subjected to heat and smoke. “Results of this study indicated that smoke constituents react with the functional groups of meat proteins.” (Randall, 1970)  These results seem to support a reddening step before smoke is applied due to the fact that heating would release the sulfhydral groups and smoke would reduce the pH.  (Reaction sequence)


With our consideration of smoking on meat, we have also entered the discussion of the effect of heat on meat.  Before considering the effect of heat on the protein lets first see how the heat gets to it.

Mechanism of heat transfer

Heat is transferred during cooking through conduction, convection and radiation.  “Spakovszky and Greitzer (2002) defined conduction as ‘transfer of heat occurring through intervening matter without bulk motion of the matter,’ convection as heat transfer due to a flowing fluid, either a gas or a liquid, and radiation as ‘transmission of energy through space without the necessary presence of matter.’   Radiation can also be important in situations in which an intervening medium is present, such as heat transfer from a fire or from a glowing piece of metal (Spakovszky and Greitzer 2002).”  (Yu, T.Y., et al, 2017)

“Meat cooking usually involves more than 1 mode of heat transfer (Bejerholm and others 2014).”   During cooking in a smoke house, heat treatment is achieved through dry heat surrounding the meat, but during reddening and smoking the air is or become moist and moist-heat (hydrothermal) thermal processing uses hot steam. Smoke House thermal treatment, including smoking, is, in reality, a combination of dry heat and moist heat.  (Yu, T.Y., et al, 2017)

“Conventional cooking of meat results in heterogeneous heat treatment of the product on account of steep temperature gradients (Tornberg 2013). Emerging mild cooking technology such as ohmic cooking can achieve a more homogeneous heating by heating the entire volume of meat at the same time (Tornberg 2013).”  (Yu, T.Y., et al, 2017)

This is important in considering the effect of heat on the grid system proposed by Draven, made from perforated 304 stainless steel, 1.6mm thick with 5mm holes.   This means that on a 100mm sq area, there are 181 holes of 5 mm radius.    The hole area is then 3547 and the metal area is 6452 which gives a ratio of approx 1:2, hole to steel (exactly 1:1,8).  The exposed meat area is therefore approximately half (take the edging to be approximately 0.02 to give the total ratio of 1:2).  This amplifies the effect of heating, but by what factor?  We will determine this.

“Heat may cause proteins to lose their native conformation (denature) by providing the polypeptides with kinetic energy, increasing their “thermal motion,” and thus rupturing the weak intramolecular forces (such as nonpolar interaction, various kinds of electrostatic interaction, and disulfide bonds) that hold the proteins together (Davis and Williams 1998). As the temperature increases, a protein starts to unfold. When almost all the tertiary and secondary structures are lost, the unfolded protein may aggregate, have its disulfide bonds scrambled, undergo side-chain modifications (Davis and Williams 1998), and cross-link with other polypeptides. Aggregation is the consequence of nonpolar interaction between heat-denatured proteins whose hydrophobic groups have turned outward into the surrounding water, in order to adopt a lower energy state (Davis and Williams 1998). A variety of side-chain modifications, such as those induced by oxidation or the Maillard reaction, have been characterized in proteins following heat treatment.”  As heat increases, the 3-dimensional structure of meat proteins change.  These changes manifest in a change in colour and gelation.  (Yu, T.Y., et al, 2017)


“Upon thermal processing, globin denatures and detaches itself from the iron atom, and surrounds the hem moiety.  Nitrosylmyochromogen or nitrosylprotoheme is the pigment formed upon cooking and it confers the characteristic pink colour to cooked cured meats.”  (Pegg, R. B. and Shahidi, F; 2000: 42)


Skeletal muscles are bundles of muscle cells (also known as muscle fibers) embedded in connective tissue.  (Yu, T.Y., et al, 2017) These muscle proteins “are grouped in three general classifications: (1) myofibrillar, (2) stromal, and (3) sarcoplasmic. Each class of proteins differs as to the functional properties it contributes.”  (www.meatscience.org)

->  Myofibrillar Proteins

“Muscle fibers, the muscle cells which are grouped into muscle bundles, are composed of myofibrils. The proteins that comprise the myofibril, including actin and myosin and several more, are collectively called the myofibrillar proteins.  The myofibrillar protein components most important for muscle fiber structure are actin and myosin. They are the most abundant proteins in muscle and are directly involved in the ability of muscle to contract and to relax.”  (www.meatscience.org)

Myosin is a protein which is described as the motor, and the structural protein, actin’s filaments are the tracks along which myosin moves, and ATP is the fuel that powers movement.  (Lodish, 2000)  Myosin “converts chemical energy in the form of ATP to mechanical energy, thus generating force and movement.”  (Cooper.  2000)

-> Sarcoplasmic Proteins

“The sarcoplasmic proteins include hemoglobin and myoglobin pigments and a wide variety of enzymes.  Pigments from hemoglobin and myoglobin help to contribute the red colour to muscle.” (www.meatscience.org)

-> Stromal Proteins

“Connective tissue is composed of a watery substance into which is dispersed a matrix of stromal- protein fibrils; these stromal proteins are collagen, elastin and reticulin.

Collagen is the single most abundant protein found in the intact body of mammalian species, being present in horns, hooves, bone, skin, tendons, ligaments, fascia, cartilage and muscle. Collagen is a unique and specialised protein which serves a variety of functions. The primary functions of collagen are to provide strength and support and to help form an impervious membrane (as in skin). In meat, collagen is a major factor influencing the tenderness of the muscle after cooking. Collagen is not broken down easily by cooking except with moist—heat cookery methods. Collagen is white, thin and transparent. Microscopically, it appears in a coiled formation which softens and contracts to a short, thick mass when it is heated and helping give cooked meat a plump appearance. Collagen itself is tough; however, heating (to the appropriate temperature) converts collagen to gelatin which is tender.

Elastin (often yellow in colour) is found in the walls of the circulatory system as well as in connective tissues throughout the animal body and provide elasticity to those tissues.

Reticulin is present in much smaller amounts than either collagen or elastin. It is speculated that reticulin may be a precursor to either collagen and/or elastin as it is more prevalent in younger animals.”  (www.meatscience.org)


Solubility of the different skeletal muscles is of huge importance in meat processing.  In terms of the use of Transglutaminase, different proteins are used in the TG mix as added connective protein to enhance the overall binding action.  When TG is mixed in a solvent before application, different solvents will provide different solubility which may concern operators.

For example, TG containing stromal proteins such as collagen shows low solubility in a neutral aqueous solvent such as water but high solubility in a curing brine solution with phosphates and salts on account of the high ionic charge of this solution.

Skeletal proteins.png

From Yu, T.Y., et al, 2017.

The other important reason for solubility relates to water absorption and colour development.


“Bendall and Restall (1983) systematically studied the physical changes occurring during heating of intact beef-derived single muscle cells, and also the very small myofiber bundles of 0.19 mm in diameter (containing 40 to 50 cells) at final temperatures between 40 and 90 °C. In addition, the authors also studied heating of larger bundles of 2 mm in diameter.”  (Yu, T.Y., et al, 2017)

According to their work, the stewing process progresses as follows:

From 40 to 52.5 °C

Denaturation of sarcoplasmic (include hemoglobin and myoglobin) and myofibrillar proteins occurs.  Related to colour development the denaturation will effect sarcoplasmic protein even though its denaturation probably occurs from at least 25 °C.   Related to moisture and the range of 40 to 52.5 °C, a slow loss of fluid from the myofibers into the extra-myofiber spaces occurs without shortening. (Yu, T.Y., et al, 2017)

Between 52.5 and 60 °C.

At this temperature, there is “an increasingly rapid loss of fluid from the myofibers, reaching a maximum rate and extent at about 59 °C.”  There is no overall shortening at this temperature mainly due to heat shrinkage of the basement membrane collagen (type IV and perhaps type V as well) at about 58 °C.   (Yu, T.Y., et al, 2017)

Between 64 to 94 °C.

Considerable overall shortening and a decrease in cross-sectional area are noted, accompanied by increased cooking loss with heat shrinkage of the endomysial, perimysial, and epimysial collagen.”  (Yu, T.Y., et al, 2017)

Long Stewing.

“Long periods of stewing causes partial or complete gelatinization of the epimysial collagen, followed by the peri- and endomysial collagen, resulting in the soft and tender feature of stews (Bendall and Restall 1983). It is worth mentioning that meat with a high pH (Zhang and others 2005) or fat content (Wood and others 1986; Jung and others 2016) has been shown to exhibit higher water-holding capacity.”  (Yu, T.Y., et al, 2017)

The important aspect for us is the key temperature of < 52.5 where moisture loss becomes “rapid”.  This gives us an important upper “meat temperature” limit above which rapid moisture loss occurs.



Kajitani, et al, (2011) studied the kinetics of thermal denaturation of protein in cured pork meat related to each of the three protein classes of meat proteins namely myosin (from myofibrillar proteins), sarcoplasmic proteins and collagen (from stromal proteins).  Of great interest to us is the sarcoplasmic proteins which include the pigment containing myoglobin.

The first important consideration is that the “thermal denaturation of muscle proteins such as myosin, sarcoplasmic proteins and collagen, and actin, occurs at different temperatures. To describe those reactions during thermal processing, temperature dependencey of the reaction rate constant is necessary.”  As the level of NaCl in the meat increased, “the thermal-denaturation rate constant of each protein increased.” (Kajitani, et al, 2011)

For the purpose of this article, sarcoplasmic proteins added salt starts to denature at a temperature of around 50 deg C, reaching a peak at around 68 deg C.  Adding Sodium Chloride moves the graph to the left.


Graph source:  (Kajitani, et al, 2011)


Cooper GM.  2000.  The Cell: A Molecular Approach. 2nd edition.  Sunderland (MA): Sinauer Associates.

Kajitani, S., Mika Fukuoka, M., and Sakai, N..  2011.  Kinetics of Thermal Denaturation of Protein in Cured Pork Meat.  Japan Journal of Food Engineering, Vol. 12, No. 1, pp. 19 – 26, Mar. 2011

Lodish H, Berk A, Zipursky SL, et al.  2000.  Molecular Cell Biology. 4th edition.  New York: W. H. Freeman; 2000.

RANDALL, C. J. and BRATZLER, L. J. (1970), CHANGES IN VARIOUS PROTEIN PROPERTIES OF PORK MUSCLE DURING THE SMOKING PROCESS. Journal of Food Science, 35: 248–249. doi:10.1111/j.1365-2621.1970.tb12149.

Yu, T.-Y., Morton, J. D., Clerens, S. and Dyer, J. M. (2017), Cooking-Induced Protein Modifications in Meat. COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY, 16: 141–159. doi:10.1111/1541-4337.12243