Leghemoglobin is probably the reason why there has been such dramatic rise in the meatless or meat-free burger. Might seem odd to say but it is one of those intriguing ingredients which makes it possible to have a meat-like flavour and colour without resorting to becoming a carnivore if that is what does it for you.
The name is a shortened moniker of legume hemoglobin. It is a monomeric protein which contains a haem (or heme) group. This is the type of molecule that contains iron at its centre and allows it to reversibly bind oxygen. In blood, animal haemoglobin is essential for the transport of oxygen to all cells and for the removal of carbon dioxide. Myoglobin which is a monomer is the protein that receives oxygen from hemoglobin and resides in all muscles.
In some plants, this type of hemoprotein is regularly found in nitrogen-fixing root nodules of legumes such as soy and alfalfa where it has an important but indirect role in the nitrogen-fixing process.
The Role Of Leghemoglobin In Plants.
Leghemoglobin is only induced (i.e. produced from the genome) when the plant roots are infected by a bacterial genus called Rhizobium. There is also a protein variant which is a dimeric hemoglobin apparently that occurs in the nitrogen-fixing root nodules of Parasponia. This is a non-legume which can form nodules with Rhizobium.
The protein can bind and release oxygen molecules in a reversible manner. Leghemoglobin a shows significantly higher oxygen and carbon monoxide affinities and association rates than myoglobin (Rohlfs et al., 1988).
Nitrogen-fixing bacteria known as rhizobia form a symbiotic relationship between the bacteria associated with the plant roots. The protein reduces the presence of oxygen in root nodules so that the enzyme nitrogenase can help fix nitrogen from the atmosphere. Oxygen is an inhibitor of this enzyme. Leghemoglobin also appears to help the nitrogen-fixing bacteria by providing oxygen molecules for bacterial respiration at low partial pressures (Appleby, 1984).
Leghemoglobin (Lb) has a molecular weight of 15.9 kDa and is very similar to myoglobin in its behaviour (as we have already referenced).
The protein was first characterised by Kubo in 1939 and then leghemoglobin in 1945 (Virtanen et al., 1945). A great deal of research has been conducted on this protein by Ellfolk (1972).
Its Role In Creating Meat Flavours
The molecule not only adds a vibrant red colour but helps with creating the taste of meat. It is also a contributor to texture but not to the same extent as its other properties. The ingredient is added to vegetable protein to not only provide the colour in a meat analogue but also add that bleed and sizzle when the burger or patty is fried. The effect is very realistic.
According to Impossible Foods, the maximum rate of inclusion of soy leghemoglobin in meat analogue products is 0.8 g per 100 g of meat analogue, although typical use rates are generally around 0.45 g per 100 g of meat analogue. The heme ingredient is added after all heating processes have occurred in the production of the burger. This is to prevent the iron which is in its oxidative II state turning brown-green when it enters the III state.
At the current time of this article, there were no known identified suppliers of leghemoglobin to product developers other than those who were using classical extraction methods. The intention has in the past been to generate a small amount of material for product developers to try in meatless products. These suppliers are currently based in China and are only known to those active in producing vegetarian and vegan foods. It is known to the author that other genetically modified forms of leghemoglobin are being engineered into related microorganisms.
Stability Of Leghemoglobin In Foods
Leghemoglobin should behave like myoglobin when it is cooked in foodstuffs such as meatless burgers. A fresh looking meatless burger containing this protein actually looks relatively bright red with its colours modified by the presence of other ingredients.
Colour, when it comes to meat is of critical importance to the consumer because it not only reflects freshness but also a measure of ‘doneness’. In other words, the colour of haem for example is bright red and becomes a dullish red-brown with hint of grey. Leghemoglobin in a foodstuff conveys a sense of doneness in a similar fashion because depending on how it has been cooked there are variations throughout the meatless product which convey a similar sense.
The change in colour of myoglobin during cooking of beef for example was assessed back in the late fifties (Bernofsky et al., 1959). If you expect leghemoglobin to perform like myoglobin, these studies have considerable resonance. When beef was cooked to an internal temperature of 60°C or lower, then the meat is considered rare and a steak would have a bright red colour. Any cooking temperature between 60°C and 70°C produces a rare-medium cook and the interior would be pink. Any higher cooking temperature and the meat is well done. It looks grey to brown throughout. As with an Impossible Burger, the surface receives most of the heat and so the pigment changes throughout corresponding to the level of cook given.
Although I’ve yet to find a direct reference to this, it is assumed that nitrosating agents react with the protein. A typical and characteristic colour of cured meats comes from reactions between myoglobin and nitrite. In this example, myoglobin is oxidized indirectly using nitrite to form metmyoglobin (brown). A subsequent reaction produces a complex with nitrite that is reduced to nitric oxide myglobin which returns the red-pink colour. Leghemoglobin should behave similarly to form a variant called metleghemoglobin. The addition of erythorbate and ascorbate apparently accelerates the formation of the pink-red complex. When a cooked ham treated with nitrite is heated for example, any nitric oxide myoglobin will form a highly stable pink pigment called nitrosylhemochrome. This pigment contains two nitric oxide ligands. The issue might present a similar safety one for leghemoglobin if nitrite is used in any way (Barnett & King, 1997).
Sulphites probably also reacts similarly with leghemoglobin as it is known to do with myoglobin. Sulphites are banned in meat in the USA because sulphite forms an S-heme complex with both myoglobin and hemoglobin. It should have the same bright red colour as oxyhemoglobin. It could be used as an adulterating agent if misused (Barnett & King, 1997).
The role of pH will presumably be important, it certainly has an impact on myoglobin protein conformation which controls access to the haem binding site. The effect is most marked by the rate of autooxidation. When the globin (protein) part is denatured as in cooking, it becomes more prone to autoxidation. Acidification of meat for example speeds up the rate of autooxidation and the effect is likely to occur with leghemoglobin.
Myoglobin stability is improved by the use of vitamin E and other tocopherols such as alpha-tocopherol. Preventing the leaching of iron is important. Leghemoglobin will bind carbon monoxide and carbon dioxide (Morikis and Wright, 1996).
Health & Safety
Impossible Foods have lodged their requests with both FDA and EFSA so that it can be permitted for food use in the future. So far, nearly 20 million vegan burgers containing what is defined as a meat analogue have been sold in the USA since 2016. In addition to over 11,000 restaurants in the U.S.A., at least 300 restaurants in Hong Kong and Macau have also offered meat analogue products containing soy leghemoglobin without any evidence or reports of any safety issues concerning this ingredient, indicating that the meat analogue products containing LegH Prep are well tolerated.
The haem B molecule in leghemoglobin is structurally very similar to myoglobin and is a source of iron. Thee is a direct claim that the amount of haem iron is comparable to an equivalent serving of a corresponding animal-derived meat product. In their application information to EFSA, by way of an example
“…each 113 g serving of the Impossible™ Burger will provide approximately 4.2 g of total iron, of which 1.8 mg is the haem iron contributed by the soy leghemoglobin.”
The comparison is made with an equivalent serving of ground or minced beef of 113 grams containing 80% lean meat and 20% fat which contains approximately 2.2 mg of total iron (USDA, 2019). It is assumed that approximately 70 to 90% of the iron in beef is in the haem iron form. So a 113g serving of ground beef provides 1.5 to 2.0 mg of haem iron. This is comparable to the amount of haem iron coming from soy leghemoglobin.
The most potent story at the moment is regulatory. One of the issues is that there is a version from Impossible Foods (USA). It is genetically engineered because it is produced in the microbial cells of a Pichia yeast species. That means it can be produced in greater amounts and in controlled conditions. This variant can only be used by Impossible Foods and they will not allow its use by any other manufacturer. The issue is that not all businesses want GMO (Genetically Modified Organism) ingredients and some have an active policy to reject any ingredient which is GMO.
The host yeast, Pichia pastoris is nontoxigenic and nonpathogenic and has been used in the recombinant expression of both Generally Recognized As Safe (GRAS) and US Food and Drug Administration (FDA)-approved proteins. In the fermentation process, there are a number of other proteins released which may or may not be part of the leghemoglobin.
The FDA approved the use of soy leghemoglobin in July 2019. It received a green light from the FDA for its use as a colour additive. It can only be used as a colour additive in ground beef analogue products but the amount of this protein must not exceed 0.8 per cent by weight of the uncooked product. as a colour additive it will achieve its intended technical benefit. The value is based on the range for myoglobin in meat muscle – in beef this is between 0.8% and 1.8% w/w. The level is also set because any higher produces unacceptable flavour issues. The FDA has amended its own colour additive regulations to provide for the safe use of leghemoglobin.
At the end of 2019, the USA’s Food And Drug Administration (FDA) rejected objections made by the Center for Food Safety concerning the safety of genetically engineered soy leghemoglobin. For some vegans the use of soyhemoglobin is an issue because substantial animal testing was required to validate the safety of the ingredient.
One business in particular, Impossible Foods, Inc. is using soy leghemoglobin in its vegan burgers. There are already on sale in the USA in various types of outlet. The Impossible Burger also employs other genetically modified ingredients such as soy protein. At the moment the main safety testing has been with rat feeding studies from which there have been no identified side effects.
Impossible Foods filed an application in October 2019 in the European Union for the use of leghemoglobin. European regulations covering the use of genetically modified foods are probably some of the strictest in the world. Regulation (EC) No. 1829/2003 states that for any genetically modified food or feed to the enter the supply, the GMO must be approved by the EC following a safety assessment by the European Food Safety Authority. The evaluation is performed by the Standing Committee on the Food Chain and Animal Health before a GM crop say is cultivated or a GM food ingredient is introduced.
The request covers application of soy leghemoglobin in all food and feed uses but excludes cultivation of the protein in the EU. All the ingredient will come from outside the EU.
The European Parliament has been very clear to state that on the packaging, the label “This product contains genetically modified organisms” or “the product contains genetically modified [name of organism]” directly on the label. Likewise, if the ingredient is derived from a GMO it must be stated. Under EU regulations it will probably need to have an E number if its is classified as a food colour or flavour because it is an additive.
Whilst there are no GMOs in the final ingredient, because the yeast is removed on harvesting the heme protein, the end product in the USA for example has not been able to qualify of the coveted Non GMO Project Verified stamp.
Likewise, in January 2020, Food Standards Australia New Zealand (FSANZ) has requested comments on an application by Impossible Foods Inc. to amend the Australia New Zealand Food Standards Code so that it can use the ingredient in its meat products for sale in the these Oceanic countries. This is the first call for submissions on the application, with a second round of public consultation undertaken in 2020. All FSANZ decisions on applications are notified to ministers responsible for food regulation, who can request a review or agree that the standard should become law.
Triton Algae Innovations which is based in San Diego, USA is producing the heme from an algae called Chlamydomonas reinhardtii which produces heme in the same pathway as chlorophyll. The heme is produced by shining UV light onto the algae which then produces the heme that can then be extracted and used subsequently in formulations.
Agency Response Letter GRAS Notice No. GRN 000204. (2006) United States Food and Drug Administration, Center for Food Safety and Applied Nutrition, Office of Food Additive Safety; 2006.
Ahmad, M., Hirz, M., Pichler, H., Schwab, H. (2014) Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production. Appl. Microbiol Biotechnol. 98(12) pp. 5301–5317
Appleby, C. A. (1984) Annu. Rev. Plant Physiol. 35, pp. 443-478
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Jin, Y., He, X., Andoh‐Kumi, K., Fraser, R. Z., Lu, M., & Goodman, R. E. (2018). Evaluating potential risks of food allergy and toxicity of soy leghemoglobin expressed in Pichia pastoris. Molecular Nutrition & Food Research, 62(1), 1700297 (Article)
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