Soy leghemoglobin (Glycine max legHb)

Description

• Heme-carrying globular protein naturally found in soy root nodules, where it facilitates oxygen transport in nitrogen-fixing symbiosis. In food use it is produced by fermentation, not harvested from roots.

• Structure/function: a ~15–18 kDa monomeric globin holding heme b (protoporphyrin IX–Fe²⁺). It provides raw-meat redness and cooked-meat browning, and acts as a flavor catalyst in Maillard/lipid reactions, unlocking meaty, savory, roasted notes.

• Use levels (typical): about 0.1–0.8% in plant-based meat formulas (as soy leghemoglobin preparation, which includes the purified protein and minor co-constituents from the production organism).

Key constituents

• Protein: soy leghemoglobin (legHb) with high heme occupancy.

• Heme b: responsible for color and redox catalysis; converts ferrous → ferric with heat/oxygen, shifting red → brown.

• Carrier matrix (in preparations): small amounts of peptides, carbohydrates, minerals from the production yeast and process aids (spec-dependent).

Production process

• Gene sourcing & design: the soy legHb gene is codon-optimized and inserted into a food-grade yeast (commonly Pichia pastoris / Komagataella phaffii) using GMO molecular biology.

• Fermentation: aerobic fed-batch with controlled pH, dissolved O₂, and temperature to maximize heme-loaded legHb.

• Harvest & purification: cell disruption → clarification/filtration → chromatography or polishing to spec; alternatively, a standardized legHb preparation is produced without exhaustive isolation.

• Stabilization & packaging: adjustment to target protein/iron content; pasteurization or sterile filtration; fill in oxygen-barrier packs (often refrigerated/frozen).

Sensory and technological properties

• Coloring: delivers bright red at low/intermediate pH; browns on heating like myoglobin in meat.

• Flavor catalysis: accelerates Maillard and lipid-derived pathways, enhancing umami, meaty, grilled notes at low dose.

• Compatibility: works in protein matrices (soy/pea/wheat), emulsions, and gels; activity influenced by pH, antioxidants, chelators, and metal ions.

• Heat behavior: retains color up to ~60–70 °C (ferrous state), then oxidizes/denatures, yielding brown cooked appearance.

Food applications

• Plant-based meats: burgers, minced fillings, sausages, meatballs—provides raw look, cooking transition, and meat-like flavor.

• Culinary bases: gravies, demi-glace analogs, RTS sauces, savory snacks for roasted/meaty top-notes.

• R&D uses: micro-dosed as a reaction flavor catalyst in pilot thermal systems.

Nutrition and health

• Heme iron: present as heme b with high bioavailability, but serving-level iron is modest due to low use rates.

• Protein intake: contribution is small in finished foods (sub-percent inclusion).

• Allergenicity: the legHb sequence is not a common soy allergen; however, it is a soy-derived protein and products often contain other soy ingredients—follow allergen labeling rules. Residual yeast proteins are controlled by specs and safety assessments.

• Digestibility/toxicity: safety evaluations have reported no genotoxicity, no systemic toxicity, and typical protein digestion under simulated GI conditions (jurisdictional dossiers).

Quality and specifications (typical topics)

• Identity/purity: legHb content to target; heme occupancy high; total iron within spec.

• Chemical limits: moisture, ash, pH, residual DNA, residual solvents (if any), heavy metals within limits.

• Microbiological: pathogens absent/25 g, low TAMC/yeasts/molds; endotoxin monitored for concentrates.

• Functional tests: color metrics (CIELAB a*), heme stability, thermal color change curve, and flavor potency in a model system.

• Oxidation control: low peroxides/TBA; protect from light/O₂.

Storage and shelf life

• Cold-chain recommended: 0–4 °C for refrigerated liquids; ≤−18 °C for frozen concentrates.

• Packaging: light- and oxygen-barrier containers; minimize headspace (nitrogen flush).

• Shelf-life: formulation- and pack-dependent; color/flavor potency declines with heat/oxygen exposure.

Safety and regulatory

• Regulatory status varies by region. In several jurisdictions legHb (or soy leghemoglobin preparation) has been evaluated as a color/flavor ingredient and authorized for specified uses and levels. Other regions treat it as novel food or color additive, requiring pre-market authorization.

• Labeling: declare “soy leghemoglobin” or “soy leghemoglobin preparation” per local naming rules; apply allergen and GMO/bioengineered disclosures where required.

• Exposure margins: product specifications and intended use levels are designed to keep dietary exposure well below safety thresholds established in risk assessments.

Troubleshooting

• Dull/brown color in raw matrix: excessive oxygen/oxidants, high storage T, or metal contamination → add antioxidant system (e.g., ascorbate/chelator), improve O₂ barrier, avoid Cu/Fe contact.

• Metallic/off aromas on cooking: lipid oxidation catalyzed by heme → tighten oil quality, add ROO scavengers, optimize thermal profile.

• Inconsistent redness batch-to-batch: check heme occupancy, pH, ionic strength, and dosage uniformity.

• Poor flavor impact: insufficient reducing sugars/amino donors → adjust matrix for Maillard precursors or tweak water activity/salt.

Sustainability and supply chain

• Fermentation-based heme reduces reliance on animal sources, enabling lower-GHG meat analogues.

• Implement heat/air recovery in fermentation and CIP water reuse; manage effluents toward BOD/COD targets; use recyclable oxygen-barrier packs; maintain GMP/HACCP and traceability.

Labelling

• Name: “soy leghemoglobin” (or “soy leghemoglobin preparation”).

• Include any processing qualifiers (e.g., fermentation-derived), bioengineered/GMO statements where applicable, and allergen declarations consistent with the full formula.

Conclusion

Soy leghemoglobin is a fermentation-derived heme protein that brings authentic meat color and meaty flavor generation to plant-based foods at very low doses. Success in formulation hinges on oxygen/light control, appropriate matrix chemistry for Maillard reactions, and regulatory-compliant labeling—yielding products that look, cook, and taste closer to meat.

Mini-glossary

• Heme b: the iron–porphyrin cofactor that gives meat its red color and participates in redox chemistry.

• Maillard reaction: heat-driven reaction between reducing sugars and amino compounds that creates browned colors and roasted flavors.

• Pichia pastoris / Komagataella phaffii: a yeast widely used for recombinant protein production.

• GMO/bioengineered: produced using genetic modification; disclosure and approval requirements are jurisdiction-specific.

• GRAS / novel food / color additive: common regulatory categories under which soy leghemoglobin may be assessed, depending on the country.

• NOAEL/ADI: toxicology benchmarks (no-observed-adverse-effect level / acceptable daily intake) used in risk assessment.

• BOD/COD: biochemical/chemical oxygen demand—key wastewater impact metrics.

• GMP/HACCP: good manufacturing practice / hazard analysis and critical control points, preventive food-safety systems.

References__________________________________________________________________________

Fraser RZ, Shitut M, Agrawal P, Mendes O, Klapholz S. Safety Evaluation of Soy Leghemoglobin Protein Preparation Derived From Pichia pastoris, Intended for Use as a Flavor Catalyst in Plant-Based Meat. Int J Toxicol. 2018 May/Jun;37(3):241-262. doi: 10.1177/1091581818766318.

Abstract. The leghemoglobin protein (LegH) from soy ( Glycine max) expressed in Pichia pastoris (LegH preparation, LegH Prep) imparts a meat-like flavor profile onto plant-based food products. The safety of LegH Prep was evaluated through a series of in vitro and in vivo tests. The genotoxic potential of LegH Prep was assessed using the bacterial reverse mutation assay (Ames test) and the in vitro chromosome aberration test. LegH Prep was nonmutagenic and nonclastogenic in each test, respectively. Systemic toxicity was assessed in a 28-day dietary study in male and female Sprague Dawley rats. There were no mortalities associated with the administration of LegH Prep. There were no clinical observations, body weight, ophthalmological, clinical pathology, or histopathological changes attributable to LegH Prep administration. There were no observed effects on male reproduction in this study, but the suggestion of a potential estrous cycle distribution effect in female rats prompted a second comprehensive 28-day dietary study in female Sprague Dawley rats. This study demonstrated that female reproductive parameters were comparable between rats treated with LegH Prep and concurrent control rats. These studies establish a no observed adverse effect level of 750 mg/kg/d LegH, which is over 100 times greater than the 90th percentile estimated daily intake. Collectively, the results of the studies presented raise no issues of toxicological concern with regard to LegH Prep under the conditions tested.

Bae HN, Kim GH, Seo SO. Secretory Production of Plant Heme-Containing Globins by Recombinant Yeast via Precision Fermentation. Foods. 2025 Apr 20;14(8):1422. doi: 10.3390/foods14081422. 

Abstract. Leghemoglobin (LegHb) is a plant-derived heme-containing globin found in the root nodules of legumes like soybean that can be used as a food additive for red color and meaty flavor as a plant-based meat alternative. However, conventional extraction methods face challenges of low yield and high costs. To address this issue, precision fermentation with recombinant microorganisms has been applied for the sustainable large-scale production of plant leghemoglobins. This study attempted the production of plant legHbs using recombinant yeast strains, Saccharomyces cerevisiae and Komagatella phaffii. The plant legHb genes were identified from the genome of legumes such as soybean, chickpea, mung bean and overexpressed in yeast via extracellular secretion by the signal peptide and inducible promoters. Subsequently, hemin as a heme provider was added to the fermentation, resulting in increased levels of plant legHbs. In S. cerevisiae, gmaLegHb expression reached up to 398.1 mg/L, while in K. phaffii, gmaLegHb showed the highest production level, reaching up to 1652.7 mg/L. The secretory production of plant legHbs was further enhanced by replacing the signal peptide in the recombinant yeast. The secreted plant legHbs were purified by His-Tag from a culture supernatant or concentrated via precipitation using ammonium sulfate. These results suggest that the production of plant legHbs is significantly influenced by hemin and signal peptide. This study successfully demonstrates the production of the various plant legHbs other than soy legHb that can be used as natural colors and flavors for plant-based meat alternatives.