Restructuring of whole muscle meat with Microbial Transglutaminase – a holistic and collaborative approach. (30/May/2013)
By: Eben van Tonder
15 January 2017
Follow up article: Factors Affecting Colour Development and Binding in a Restructuring System Based on Transglutaminase.
The articles on the complete bacon production system are available in booklet form: https://tgrestructuringofmeat.pressbooks.com
INDEX
HOW DOES IT WORK: THE MECHANISM OF GLUING PROTEINS
CHARACTERISTICS OF MICROBIAL TRANSGLUTAMINASE
BENEFITS: SPECIFIC APPLICATIONS
TRANSGLUTAMINASE IN COMBINATION WITH OTHER INGREDIENTS
SUMMARY
Microbial Transglutaminase (MTG) is one of the most important developments in the meat industry, comparable with the direct application of sodium nitrite to curing brines early in the 1900’s. (The Naming of Prague salt)
The meat industry has a long history of restructuring or reshaping large muscles by using various forms of technology such as casings, netting, and ham moulds. Microbial Transglutaminase (MTG) allows the meat processor the use of a natural process in conjunction with a lightweight mould or grid basket system to re-shape large meat cuts to fit into packaging, high-speed slicers, optimise the use of check weighers, materially improve slicing yields (measured as the percentage of product presented to a slicer that is packed as final product) as well as offers a substantial improvement in overall product quality.
We developed a complete system based on the use of MTG that fits its characteristics and optimises it for both a high throughput factory as well as for small processing operations and optimise the technology in full.
Matters to be considered are the nature of Microbial Transglutaminase (MTG) and its relationship with other functional ingredients, its thermal characteristics, limits on the application, an optimal and flexible process flow, possible negative effects on micro and unforeseen beneficial or negative results.
The focus is on smoked and cured products, even though the application is much wider. Anybody who is interested in joining this effort or contribute in any way to this work can contact me at ebenvt@gmail.com.
INTRODUCTION
Transglutaminase functions as a biological glue. It is important in cell-matrix interaction and the general maintenance of tissue integrity through the creation of isopeptide bonds (inducing the process of cross-linking) between Glutamine and Lysine residues when the protein molecule is enriched with this amino acid. Extensive work has been done on the benefit of such functionality in the restructuring of meat trim, but few formal studies focused on its benefit of restructuring large muscle pieces such as shoulders or loins.
This is a short summary of the result of our work so far. Regular updates will be published. The last date of updating the work will always appear next to the title at the top of the page.
It is instructive to see, in the first place, that the enzyme is widely distributed in nature. It occurs naturally.
NATURES BIOLOGICAL GLUE
Transglutaminase is an enzyme, involved in many “glueing” activities. It is present in most animal tissues and body fluids, “and are involved in several biological processes, including blood clotting, wound healing, epidermal keratinization, and stiffening of the erythrocyte (red blood cell) membrane (Aeschlimann and Paulsson 1994). A typical example of a Transglutaminase-catalyzed protein crosslinking reaction is the blood coagulation when a wound heals by something called Factor XIIIa which is an activated form of plasma Transglutaminase.” (Yokoyama, K., et al.. 2004.)
Animal transglutaminases are involved in physiological processes. Other forms of transglutaminases are found in plants with various types of functioning in one plant or even in one organelle. Here, enzymes play a role in plants’ processes of growth and development. One interesting example of a feature of plant transglutaminase enzyme is its sensitivity to light. This property applies, especially to chloroplast transglutaminase. (Kieliszek, M and Misiewicz, A.; 2013)
This already points to many possible applications in meat processing such as its involvement in the stabilisation of the muscle matrix and adhesion protein. (Griffin, M., et al.; 2002) It is widely distributed in nature and should dispell the myth that by using this in meat processing, anything unnatural is being done or used. This is a natural process.
HOW DOES IT WORK: THE MECHANISM OF GLUING PROTEINS
Transglutaminase is an enzyme (in mammals, it is Calcium-dependent) that catalysis several acyl transfer reactions. (Keillor, J. W et al. 2014) The glutamine residue of a protein acts as the acyl donor and either water or a primary amine (for example Lysine) becomes the acyl acceptor.
Enzymes showing transglutaminase activity is widespread in nature. Soil bacteria, plants, invertebrates, amphibians, fish and birds. (Griffin, M., et al., 2002) Transglutaminase functions as biological glue in two important ways. On the one hand, it is important in cell-matrix interaction and the general maintenance of tissue integrity through the creation of isopeptide bonds (inducing the process of cross-linking) between Glutamine and Lysine residues when the protein molecule is enriched with this amino acid. In this case, a primary amine is the acyl acceptor of this amino acid.
The transfer of acyl onto a lysine residue bound in the polypeptide chain induces the process of cross-linking, i.e. the formation of inter- or intramolecular cross-links ε-(γ-Glu)Lys (Kieliszek, M and Misiewicz, A. 2013).
On the other hand, it plays an important role in cell death as it speeds up the chemical reaction (catalyses) of the breakdown of amino acids (deamination) “if there is an absence of free amine groups. In this case, water acts as an acyl acceptor (Motoki and Seguro 1998; Kuraishi et al. 2001). The reactions that are catalysed by this enzyme result in significant changes in the physical and chemical properties of proteins, such as modifications in viscosity, thermal stability, elasticity and resilience of proteins.” (Kieliszek, M & Misiewicz, A.; 2013) This will also happen in the presence of excess transglutaminase.
HISTORY
Clarke et al. introduced the term transglutamine in 1957 to describe the transamidating activity observed in guinea-pig liver. Transamidating is the transferal of an amide group from one compound to another. Amides (RC(O)NR2) and esters (RC(O)OR’) are classes of acyl compounds. (Griffin, M. et al.; 2002)
“Later studies undertaken by Pisano et al., on the stabilization of fibrin monomers during blood clotting, demonstrated that transamidation (transferal of an amide group from one compound to another) is brought about by enzymes which cross-link proteins through an acyl-transfer reaction between the γ-carboxamide group of peptide-bound glutamine and the ε-amino group of peptide-bound lysine, resulting in a ε-(γ-glutamyl)lysine isopeptide bond (Griffin, M. et al.; 2002)
It was known that a similar enzyme plays a role in molding fish protein pasts into Kamaboko, a popular Japanese dish. A food research group at Ajinomoto Co. “confirmed rapid gelation of several food protein solutions by the enzyme obtained from guinea-pigs liver, which led to the following development of the microbial transglutaminase jointly with Amano Pharmaceutical Company.” (Fiechter, A.; 2000: 57)
During the 1980’s Yokoyama, Nio and Kikuchi from Ajinomoto were involved “in investigating the feasibility of modifying food protein in industrial applications using the guinea pig liver enzyme and they used whey proteins and actomyosin from beef, pork, chicken or fish as substrates that could be gelled. Subsequently, improvements in the solubility, water-holding capacity and thermal stability of food proteins were demonstrated” (Yokoyama, K., et al.; 2004) which today forms the basis of its application in meat processing.
The use of guinea pig livers was however unacceptable for use in food manufacturing and it hindered its commercialization. The critical issue was the mass production of transglutaminase. (Yokoyama, K., et al.; 2004) Yokoyama, Nio, and Kikuchi from Ajinomoto, in collaboration with Amano Enzyme Co. (Nagoya, Japan) set out to find a constant supply of transglutaminase. In the process, they screened around 5,000 microorganisms for transglutaminase “and identified some microorganisms that produce TGase-like enzymes using the hydroxamate assay (Ando et al. 1989). These microorganisms excreted the enzyme, and one of them produced a high activity. The enzyme in the latter strain was shown to form G-L bonds in proteins, the critical property of a Transglutaminase (Nonaka et al. 1989); it was named microbial transglutaminase (which I abbreviate as MTG), and the source was classified as a variant of S. mobaraensis (Washizu et al. 1994).” (Yokoyama, K., et al.; 2004)
The enzyme is capable of gelling concentrated solutions of proteins such as soybean protein, milk proteins, and gelatin and myosin of various origins to produce gels with novel physical properties. The enzyme also causes crosslinking of two or more different proteins to produce new protein conjugates with novel functions. (Fiechter, A.; 2000: 57) The same microbial transglutaminase was later isolated in Physarum polycephalum and in Bacillus subtilis spores. (Kieliszek, M and Misiewicz, A.; 2013)
In mammals, transglutaminase requires Calcium. “Shimba et al. (2002); Washizu et al. (1994) and Ando et al. (1989) found that transglutaminase isolated from Streptoverticillium mobaraense did not require calcium ions” (Kieliszek, M and Misiewicz, A.; 2013)
The properties of microbial transglutaminase, its ability to cross-link most food proteins, find tremendous application in binding meat together in the process of restructuring. It is also known to dramatically increases the ability of proteins for emulsification with great impact on the use of soy proteins for example in sausage production. (Fiechter, A.; 2000: 57) It improves muscle texture and therefore product quality and assists in the reshaping of large muscles.
CHARACTERISTICS OF MICROBIAL TRANSGLUTAMINASE
“Microbiological transglutaminase is a single polypeptide with a molecular weight of approx. 38 kDa. It is composed of 331 amino acids, with an isoelectric point at pH 8.9. It is a simple monomeric protein (not a glycoprotein or lipoprotein). (Kieliszek, M and Misiewicz, A.; 2013
“A temperature of 40 °C at pH 5.5 is the most favourable for the catalytic activity of transglutaminase, with the exception of transglutaminase isolated from Streptomyces sp., which acts most effectively at a higher temperature of 45 °C. This enzyme is not stable at 50 °C (since it loses 50 % of its activity when heated for 30 min) and is very susceptible to heat in the presence of ethanol. (Kieliszek, M and Misiewicz, A.; 2013)
Smoking – keep core temperature > 40 deg C and < 50 deg C.
This temperature range is very fortunate for bacon producers since adverse visual changes start happening at around 51 deg C in meat. The subject of the denaturing of proteins is a bit more complex than one may think. Generally “thermal denaturation of muscle proteins such as myosin, sarcoplasmic proteins and collagen, and actin, occurs at different temperatures. ” (KAJITANI, S., et al; 2011) Due to the importance of this topic, it will be considered in a separate article, attached to this discussion article.
The addition of carbohydrates, such as maltodextrin, saccharose, mannose, trehalose and reduced glutathione (GSH), significantly increases the thermal stability of the enzyme. Casein may protect transglutaminase against degradation by extracellular proteolytic enzymes. At temperatures close to 0 °C, transglutaminase maintains its total enzymatic activity.” (Kieliszek, M and Misiewicz, A.; 2013)
Micro is key in factory temp. We can keep it cool! No problem for TG!
“Enzymes biosynthesised by bacteria are stable at a wide range of pH values, i.e. from 4.5 to 8.0. In addition, they do not require calcium ions to be activated, which is in contrast to transglutaminases of animal origin. This is a highly desirable property, from a practical point of view, for use in enzymatic preparation. The activity of transglutaminase increases in the presence of Co2+, Ba2+ and K+.” (Kieliszek, M and Misiewicz, A.; 2013) We will exploit this feature by designing a complimentary injection brine.
Microbial transglutaminase is inhibited by Zn2+, Cu2+, Hg2+ and Pb2+ ions which bind to the thiol group of cysteine in the active centre. (Kieliszek, M and Misiewicz, A.; 2013) We will evaluate the implications by nalizing our water and salt.
Determine through experiment the impact of dissolved potassium chloride on the functionality of TG.
Due to the importance of this topic will be considered in a separate article, “Curing Brine and Microbial Transglutaminase (MTG) – designing the optimal blend”
BENEFITS: SPECIFIC APPLICATIONS
The application in the meat industry is enormous. Costs are reduced dramatically and quality is improved significantly. In our experience, no single another technology offers this level of positive impact both for the producer and the consumer.
In terms of whole muscle meat, the application is to reshape it into a regular structure.
It is very important to understand that reshaping does not only involve MTG, but critically, also a ridged structure to create the shape and freezing. The shapes are created in this system through the use of specially designed lightweight grid baskets by an engineering company. Design considerations were ease of handling, efficiency, and cost.
The five areas where processors report the most pronounced benefits from the total system are:
1. Slicing yields
At our own plant slicing yields with the use of this system is between 90% and 95%. It has been reported in private communication from other production managers, that they regularly achieve slicing yields of 98% by a consistent application of this overall system.
2. Production yields
A reduction of losses during thermal processing. When we do not use this system, losses during smoking is between 8% and 12%, depending on the meat quality, the cut, the initial injection rate and tumbling time. When the system is used, with all other factors remaining the same, moisture loss during smoking is reduced to between 2.5% and 5%.
3. Improved product quality
Product quality improves materially in terms of juiciness, overall texture, and colour development. Catering and food services clients add the benefit of consistent product portioning.
These changes are attributed to a variety of factors in combination. Pressure is added to the meat when they are places in the grids baskets. The effect of such pressure is the current subject of several studies and seems to be playing a bigger role than originally thought.
A second reason is the effect of MTG. A third reason is that a specially coated, smoke-permeable paper is wrapped around the meat cut before it is placed inside the grid basket. This retards the unnecessary loss of moisture, especially at the meat surface during thermal processing and it distributes the heat better.
The orientation of the meat during thermal processing plays a huge roll. Traditionally such cuts are hung on smoker trolleys which favour the release of moisture due to the pressure matrix that develops inside the muscles. Laying the meat down in a trolly materially alters this pressure matrix and substantially reduce unnecessary moisture loss which impacts both production cost and product quality.
What is the exact contribution of each of these factors in isolation? How can ham technology be incorporated into bacon production?
4. New products
For the producer, Transglutaminase further allows for the utilisation meat offcuts and by-products, such as collagen, blood proteins and mechanically deboned meat, in manufacturing meat products with a higher nutritive value by supplementing it with amino acids in which it is deficient (e.g. exogenous lysine). (Kieliszek, M and Misiewicz, A.; 2013) Ultimately the consumer wins through the lower price point of these products. Generally, opportunities exist in the production of fine and coarse-minced sausages, Vienna sausages and smoked meat. Instead of high-quality meat, lower quality raw materials and additives, such as skimmed milk powder, soy or wheat flour, can now be used to manufacture products. (Kieliszek, M and Misiewicz, A.; 2013)
5. R&D component
In the work that we do here, a very strong R&D component comes with the Grid system, the Transglutaminase and how the entire process is put together. Finding new and innovative ways of exploiting any new system is of tremendous benefit. Networking makes this task easy and this brings to the fore the power of this forum.
I report on these “war stories” in Applications and Clever Ideas
The overall system as utilised in our facility is the following.
System: Inject with a multi-needle injector -> tumble under vacuum -> fill into lightweight, specially designed moulds or grids -> thermal treat/ smoke -> chill -> freeze -> slice. Sanitise – repeat.
System benefits: Ensure * ease of handling both filling and closing the lid without specialised equipment needed; * effective for small processors and large plants; * modular.
Filling the grids: A simple system has been designed to facilitate filling the grids.
The features and benefits of the system are in our view unparalleled in terms of recent developments in food science.
TRANSGLUTAMINASE IN COMBINATION WITH OTHER INGREDIENTS
We evaluate the composition of a mix of ingredients that complements transglutaminase applied in a meat processing and curing environment. The enzyme is required in very small quantities in meat processing. Experience has shown that it is best used along with a carrier that makes it more manageable as an ingredient on its own. Secondly, when you are binding meat together with TG, then you need a connective protein.
Meat – Protein – Meat
Let’s look at some options.
GELATIN OR COLLAGEN
The first such ingredient is gelatin or collagen which is a such a connective protein. It is a structural protein that imparts rigidity. It is made up of amino acids which comprise approximately 30% of the proteins within the body. They represent rigid structures, found in bones, tendons, and ligaments. In the skin, it provides firmness, suppleness and constant renewal of skin cells. It is responsible for skin elasticity. (Dr Ananya Mandal)
Gelatin is the heat-denatured, partially hydrolyzed form of native, insoluble collagen. The temperature at which the collagen protein unfolds (denature) depends on the species and hydroxyproline content. The unraveling of the collagen protein results in a viscous colloidal solution of gelatin. (Tarté, R.; 2009: 152)
Gelatin is in the first place a “thickening agent” with very good thermal stability. Its value in meat processing where primals are injected is in the first place not in its ability to form cross-links through transglutaminase, but to thicken inside the meat before the enzyme had a chance to create the cross-links and as a result of this, to minimize syneresis of the injection brine after the tumbling phase under vacuum.
Gelatin is the ideal thickening agent to accompany transglutaminase since it contains a variety of different amino acids, including our old friends Glutamine and Lysine which are now cross-linked by the action of transglutaminase. (Aguilar, M. R. and Román, J. S.; 2014: 186) It is important to use the right kind of gelatin. Fish and pork gelatin will be objectionable for either religious or allergen concerns by various processors in various parts of the world and it is an important consideration.
Why will collagen be preferred in certain conditions as opposed to gelatin in others? Which condition will favour which application?
Is there a way to ensure maximum “retention” of TG within the meat matrix.
I.e. purge after tumbling may reduce the efficacy of the TG. What hydrocolloids can possibly perform better in conjunction with a connective protein?
I recently learned about new technology that facilitates cold gelling when the brine enters the meat matrix. How can this be used in combination to achieve greater retention of brine after tumbling and, at the same time, works well in combination with TG and collagen?
The second functional ingredient that becomes important in administering transglutaminase is polyphosphate.
PHOSPHATES IN THE BRINE
(point to be re-visited in its entirety)
Adding phosphates to the transglutaminase/ gelatin mix raises the pH of the solution so that transglutaminase does not begin to set while in solution. As soon as the mix touches the substrate of the meat, the pH is lowered and the enzyme becomes active. Phosphates are, however, not the only way that this can be achieved and a new formulation is currently being designed and tested by myself and various collaborators which will exploit other systems to achieve the same results. The goal in this particular work is to design a phosphate free brine to be used in conjunction with the MTG.
Due to the importance of this topic will be considered in a separate article, “Curing Brine and Microbial Transglutaminase (MTG) – designing the optimal blend”
INJECTION BRINE
From the perspective of MTG, it is useful to add a small amount of potassium salt to the brine mix. There is an opportunity to re-design the brine system.
Due to the importance of this topic will be considered in a separate article, “Curing Brine and Microbial Transglutaminase (MTG) – designing the optimal blend”
MALTODEXTRIN
A further important ingredient is maltodextrin.
Maltodextrin is a bulking agent produced from starch and consists of beta-D-glucose. (Kerry, P. and Kerry, J. F.; 2011: 262) “Glycerol, maltodextrin, sorbitol, and xylitol are often used to preserve proteins and enzymes.” “Glycerol and maltodextrin have long been regarded as proteins stabiliser or additives which have the ability to affect the equilibrium between the nature and the unfolded conformational state of the enzymes.
Moreover, glycerol and maltodextrin can evidently increase the viscosity of the enzyme solution, which may cause reduction of chemical or biological reaction rate which could result in inactivation of the enzymes.” (Bourneow, C., et al. 2012) An addition to these, it is the “carrier” for transglutaminase. (Kerry, P. and Kerry, J. F.; 2011: 262) It prevents clotting up of the ingredient mix.
There are other “ingredients” that comes from processing steps.
VACUUM TUMBLING
Vacuum tumbling is very important means of getting rid of air bubbles that result from mixing the solution. The surface tension of the bubbles interferes with the formation of the chemical bonds. Extracting air bubbles from the solution and the meat is important for adhesion.
TIME
It is has been suggested by some that the minimum time required for the formation of bonds is anywhere from 4 to 6 hours.
In reality, it has been shown that effective bonds from as low as three hours. More formal work on this is currently being undertaken.
PH AND TEMPERATURE
When activity is measured using synthetic substrates, Transglutaminase shows high activity in a wide pH-range, i. e. between pH-value 5 and 8. Keep the temperature between 40 and 50 deg C. For a curing and smoking processor, the denaturing of various proteins at different temperatures are important considerations from a perspective of overall colour development. It has been shown by ourselves that an internal core temperature of between 35 and 45 is sufficient to achieve more than adequate binding. This range is important since visual denaturing and a “paling effect” starts taking place from a core temperature of 51 deg C.
FREEZING
It has been found that a slightly longer time is required for freezing meat that has been processed in the overall system. The exact reasons for this are currently the subject of separate studies and will be reported on soon. Several production managers have however been interviewed and the consensus is that this increase in freezing time is marginal and is easily accommodated within current processing environment.
LEGAL ISSUES
MTG is classified as a processing aid and no labelling requirements apply to its use. This is in contrast with the use of alginate, for example. (This section to be expanded with the exact US, Canadian, EU, Chinese, Australian, New Zealand, South African, Korean and WHO classification of MTG with all legal considerations listed).
UNRESOLVED MATTERS
What is the effect of the use of transglutaminase on the overall meat environment as it affects microbial activity? Generally, our goals and priority’s are:
Achieving water salt phase of 20% or more (as it related to the moisture content in the formula (% NaCl x 100/ (% + moisture) = %); Much work on this still remains, especially in a reduced sodium environment. As I have mentioned already, focused development work is underway by myself on this and results will be made available very soon.
What exactly is the water salt phase %?
Water activity
Maintaining brine to meat ratio that will maximise anti-microbial and anti-botulinal efficacy. Meat to brine ratios of up to 20% brine to 80% meat has been used without the need for any other functionals besides sodium nitrite, sodium ascorbate or erythorbate and phosphates.
What exactly is the water activity?
CONCLUSION
One talks about a “game changer.” The discovery and commercialization of microbial transglutaminase are such a “game changer.” It is the biggest event in meat processing since Ladislav NACHMÜLLNER invented the first sodium nitrite curing brine at the beginning of the 1900’s. Over the next 50 years, it will completely transform every meat processing practice.
A multi-disciplinary approach has by far the biggest advantages in re-structuring whole muscle meats using MTG. In our experience, development work of this nature is best performed in an open forum where multiple individuals and organisations contribute to the benefit of the consumers at large.
SELECTION OF PHOTOS AND CLIPS
A few photos from our factory.
Remember to also look at the follow-up article:
Factors Affecting Colour Development and Binding in a Restructuring System Based on Transglutaminase.
(c) Eben van Tonder/ ebenvt@gmail.com
References
Aguilar, M. R. and Román, J. S.. 2014. Smart Polymers and their Applications. Woodhead Publishing.
Bourneow, C. , Benjakul, S. and H-Kittikun, A.. 2012. Impact of some additives on the stability of microbial transglutaminase from Providencia sp. C1112. As. J. Food Ag-Ind. 2012, 5(03), 226-233
Fiechter, A.. 2000. History of Modern Biotechnology I. Springer.
Griffin, M., Casadio, R., Bergamini, C. M.. 2002. Transglutaminases: Nature’s biological glues. Biochem. J. (2002) 368, 377–396 (Printed in Great Britain)
KAJITANI, S., FUKUOKA, M, 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
Karayannakidis, P. D., Zotos, A., Petridis, D., Taylor, K. D. A. 2014. The Effect of Washing, Microbial Transglutaminase, Salts and Starch Addition on the Functional Properties of Sardine (Sardina Pilchardus) Kamaboko Gels. Journal Citation Reports® (Thomson Reuters, 2015)
Kerry, P. and Kerry, J. F.. 2011. Processed Meats: Improving Safety, Nutrition and Quality. Woodhead Publishing.
Keillor, J. W., Clouthier, C. M., Apperley, K. Y. P, Akbar, A., Mulani A.. 2014. Acyl transfer mechanisms of tissue transglutaminase. Bioorganic Chemistry Volume 57, December 2014, Pages 186–197
Kieliszek, M and Misiewicz, A. 2013. Microbial transglutaminase and its application in the food industry. A review. Folia Microbiol (Praha). 2014; 59(3): 241–250. Published online 2013 Nov 8. 10.1007/s12223-013-0287-x
Dr Ananya Mandal, MD. What is Collagen? (http://www.news-medical.net/health/What-is-Collagen.aspx)
Tarté, R.. 2009. Ingredients in Meat Products: Properties, Functionality and Applications. Springer.
Yokoyama, K., Nio, N., Kikuchi, Y.. 2004. Properties and applications of microbial transglutaminase. Received: 22 August 2003 / Revised: 1 December 2003 / Accepted: 5 December 2003 / Published online: 22 January 2004; Springer-Verlag
Photo Credits:
All photo’s from http://www.ajinomoto.com/en/aboutus/history/chronology/