MEAT SUBSTITUTE COMPRISING ANIMAL MYOGLOBIN

Abstract

Described herein is a meat substitute or food ingredient comprising an animal myoglobin protein, gene construct comprising a nucleic acid encoding said protein, a host cell comprising said gene construct and a method for producing said myoglobin or said meat substitute.

Claims

1. A meat substitute or a food ingredient comprising an animal myoglobin from mammoth, pig, sheep, cow, chicken or tuna or derived therefrom.

2. A meat substitute or food ingredient according to claim 1, wherein said mammoth is a steppe mammoth or a woolly mammoth or derived therefrom.

3. A meat substitute or food ingredient according to claim 1, wherein said animal myoglobin is represented by one of the following amino acid sequences: a) comprising at least 70% sequence identity with SEQ ID NO: 3, and having at least one of the following amino acid combinations: F at position 30, and/or Q at position 65, and/or H at position 92, and/or H at position 94, and/or F at position 30 and Q at position 65, and/or F at position 30 and H at position 92, and/or F at position 30 and H at position 94, and/or Q at position 65 and H at position 92, and/or Q at position 65 and H at position 94, and/or H at position 92 and H at position 94, and/or F at position 30 and Q at position 65 and H at position 92, and/or F at position 30 and Q at position 65 and H at position 94, and/or F at position 30 and H at position 92 and H at position 94, and/or Q at position 65 and H at position 92 and H at position 94, and/or F at position 30 and Q at position 65 and H at position 92 and H at position 94; b) comprising at least 70% sequence identity with SEQ ID NO: 2 or 3, Q or H at position 65 and H at position 94 and optionally having at least one of the following amino acids at the following places within SEQ ID NO: 2 or 3: E at position 9, K at position 13, T as position 14, P at position 23, L at position 27, V at position 31, G at position 54, Q at position 65, V at position 67, Q at position 84, Q at position 88, I at position 102, E at position 123 and/or I at position 143; c) comprising at least 70% sequence identity with SEQ ID NO: 1, Q or H at position 65 and H at position 94 and optionally having at least one of the following amino acids at the following places within SEQ ID NO: 1: E at position 9, K at position 13, T as position 14, P at position 23, L at position 27, V at position 31, G at position 54, Q at position 65, V at position 67, Q at position 84, Q at position 88, I at position 102 and/or E at position 123; d) comprising at least 70% identity with SEQ ID NO: 4, 5 or 6, Q or H at position 65 and H at position 94 and optionally having at least one of the following amino acids at the following places within SEQ ID NO: 4, 5 or 6: N at position 13, Q at position 27, I at position 31, N at position 67, A at position 128, Sat position 133, A at position 145 and/or L at position 150; e) comprising at least 70% identity with SEQ ID NO: 7, Q or H at position 65 and H at position 94 and optionally having at least one of the following amino acids at the following places within SEQ ID NO: 7: Q at position 6, Q at position 10, T at position 13, I at position 14, H at position 27, Mat position 31, H at position 35, D at position 36, D at position 42, R at position 43, G at position 49, P at position 53, Q at position 55, G at position 58, A at position 67, Q at position 72, K at position 75, Q at position 79, N at position 82, S at position 85, Tat position 93, V at position 111, I at position 116, A at position 117, Eat position 118, A at position 121, S at position 128, K at position 133, S at position 145 and/or F at position 150; or f) comprising at least 70% identity with SEQ ID NO: 8, Q or H at position 65 and H at position 94.

4. A meat substitute or food ingredient according to claim 1, which does not comprise a leghemoglobin which has been produced by a bacterium living in symbiosis in the root nodules of a soy plant and/or which comprises as sole heme-containing protein the myoglobin as defined in claim 1.

5. A meat substitute according to claim 1, wherein the meat substitute mimicks the aspect, form, structure, composition, flavor, texture, color, aroma, appearance and/or nutritional value of meat.

6. A food ingredient according to claim 1, wherein the food ingredient mimicks the composition, flavor, color, aroma and/or nutritional value of meat.

7. A gene construct comprising a nucleic acid encoding a myoglobin as defined claim 1.

8. A gene construct according to claim 7, wherein the nucleic acid is selected from the group consisting of: (a) a nucleotide sequence encoding a polypeptide represented by an amino acid sequence comprising a sequence that has at least 75% sequence identity or similarity with the amino acid sequence defined in claim 3 a), b), c), or d) (b) a nucleotide sequence that has at least 60% sequence identity with the nucleotide sequence of SEQ ID NO: 9, 10, 11, 12, 13, 14, 15, 16; and (c) a nucleotide sequence the sequence of which differs from the sequence of a nucleotide sequence of (b) due to the degeneracy of the genetic code.

9. A gene construct according to claim 7, further comprising a promoter.

10. A gene construct according to claim 7, further comprising a signal peptide, preferably a signal peptide that facilitates excretion of the expressed myoglobin.

11. A gene construct according to claim 7, wherein said nucleotide sequence is represented by SEQ ID NO: 19, 20, 21, 22, 23 or 24.

12. A gene construct according to claim 11, wherein said gene construct is represented by SEQ ID NO: 25, 26, 27, 28, 29 or 30.

13. A vector comprising a gene construct as defined in claim 7.

14. A host cell comprising a gene construct as defined in claim 7.

15. A host cell according to claim 14 which is a prokaryote or a eukaryote.

16. A method for producing a myoglobin as defined in claim 1, comprising culturing the host cell of claim 14 in a suitable medium and optionally recovering the host cell and/or the myoglobin.

17. A method according to claim 16, wherein the produced myoglobin does not comprise a signal peptide.

18. A method according to claim 16, wherein the recovered myoglobin is purified, preferably substantially purified.

19. A method according to claim 16, wherein the host cell produces the myoglobin extracellularly.

20. A method for producing a meat substitute or a food ingredient as defined in claim 1, comprising formulating the myoglobin obtainable from the method of any one of claims 16 to 19 into a meat substitute or a food ingredient.

21. A protein, wherein said protein comprises a sequence comprising SEQ ID NO: 3.

Description

LEGEND TO THE FIGURES

[0381] FIG. 1. Tridimensional structure of bovine myoglobin. Structure of wildtype pork Mb, solved by X-ray diffraction at a resolution of 1.8 Å (Krzywda et al. 1998). Shown are the heme, the proximal histidine (His94) and the distal histidine (His65).

[0382] FIG. 2. Comparison of the myoglobin protein sequence from six animal species. The sequence alignment shows sequences from tuna (Thunnus orientalis), chicken (Gallus gallus), the steppe mammoth (Mammuthus trogontherii), pork (Sus scrofa), cattle (Bos taurus) and sheep (Ovis aries). The level of amino acid conservation is indicated by blue shading, using the BLOSUM62 score. The proximal histidine (His94, boxed) is present in all six species. The steppe mammoth carries a glutamine (Gln65, boxed) at the position normally occupied by the distal histidine (His65, boxed), a phenylalanine at position 30 (Phe30, boxed), and a histidine at position 92 (His92, boxed).

[0383] FIG. 3. Model of the tridimensional structure of myoglobin from the asian elephant (left) and the steppe mammoth (right). The ribbon (top) and surface (bottom) views are shown. The heme molecule is depicted, and so are the residue at position 92 (glutamine in the elephant, histidine in the mammoth). In the surface view, positive charges are shown in darker grey.

[0384] FIG. 4. Production of extracellular animal Mb in Pichia pastoris. Cell-free supernatants were collected after fermentation with (+) or without (−) methanol induction, and concentrated 10-fold by ultrafiltration. Concentrated samples obtained after methanol induction showed a dark-red color (A) and, after analysis by SDS-PAGE, revealed a protein of the expected molecular weight for Mb (B).

[0385] FIG. 5. Absence of recombinant DNA in the myoglobin preparations obtained by fermentation. The same gel is shown with two different exposure times (upper and bottom panel). The asterisk indicates the primers that remain after the PCR reaction.

[0386] FIG. 6. Sequence coverage of mammoth myoglobin identification by mass spectrometry. A bar graph (lower panel) shows the intensity of the different peptides in logarithmic scale. The sequences of the peptides at the bottom of the graph correspond to SEQ ID NOs: 31-80.

[0387] FIG. 7. Absorption spectra of mammoth and cattle myoglobin solutions. (A) The absorbance spectrum of recombinant steppe mammoth Mb (black circles), cattle Mb (white circles) and commercially available Mb, purified from equine muscle (triangles) was recorded at room temperature. (B) Absorbance spectra of horse Mb during a 24 h incubation at pH5.6 and 25° C. The insert shows the ratio between absorbance at 580 nm (MbO2) and at 505 nm (MetMb) for the recombinant mammoth Mb (black bars), cattle Mb (grey bars) and commercially available horse Mb (white bar). Asterisks represent statistically significant differences between the recombinant Mb and horse Mb: (*)=p<0.05, (**)=p<0.01.

[0388] FIG. 8. Color of lab-made meat analogues containing myoglobin. Pictures (A) and quantifications (B) showing the effect of myoglobin addition on the color of plant-based burgers.

[0389] FIG. 9. Color stability of meat analogues containing myoglobin. Pictures (A) and quantifications (B) showing color changes observed over time in plant-based burgers containing various concentrations of purified horse Mb, exposed to constant light at 4° C. A commercial burger containing soy leghemoglobin was included for comparison.

[0390] FIG. 10. Volatile compounds extracted from lab-made meat analogues containing recombinant heme proteins. (A) Quantification of the number of volatile compounds retrieved from plant-based burgers by HS-SPME GC-MS. Values not sharing the same subscript were found significantly different from each other at p<0.05 according to Tukey's HSD test. (B) Principal Component Analysis (PCA) of volatiles from lab-made plant-based burgers, either raw (shown in green) or gilled (shown in blue). A commercially available plant-based burger, containing a recombinant soy leghemoglobin (LegH), was used as reference.

[0391] FIG. 11. FIG. 11. Volatile compounds associated with the addition of recombinant myoglobin to lab-made meat analogues. (A) Relative amounts of volatile flavors active-compounds detected in grilled plant-based burgers, normalized to the levels observed in plant-based burgers without Mb. Asterisks represent statistically significant differences between the burgers without and with Mb: (*)=p<0.5, (**)=p<0.01, (***)=p<0.001. $ represent statistically significant differences between the mammoth and cattle Mb: ($)=p<0.05, ($$)=p<0.05. (B) Aroma description of the volatile compounds, which are also found in cooked meat (van Ba et al. 2021, The Good Scent Company information system).

[0392] FIG. 12. Iron bioavailability from myoglobin. Iron uptake (content of ferritin in ng, normalized to the total protein content in mg) by human Caco-2 intestinal cells exposed to purified bovine Mb at 0.5 mg/ml, 1.0 mg/ml or 2.0 mg/ml, compared to the control medium alone (CM). Asterisks represent statistically significant differences between CM and treatments: (****)=p<0.0001.

[0393] FIG. 13. Effect of recombinant myoglobin on differentiated Caco-2 monolayers. Cytotoxicity was measured after 24 h of incubation in the presence of recombinant mammoth or cattle Mb, or the complete medium (CM) as control. Asterisks represent statistically significant differences between CM and treatments: (*)=p<0.05; (**)=p<0.01.

EXAMPLES

Example 1: Production Method A

Example 1.1: General Construction of Gene Constructs and Vectors

[0394] In some instances, complete vectors and/or genomic integration cassettes suitable for expression in Pichia Pastoris, Escherichia coli and Aspergillus niger are synthetically made by commercial suppliers. In some instances, the construction of vector fragments and/or genomic integration cassettes is performed using PCR with primers designed for the introduction of compatible overhangs between fragments and commercially available polymerases, according to the manufacturer's protocol, followed by complete vector and/or genomic integration cassette assembly performed in vitro using commercially available kits, according to the manufacturers' protocols. In some instances, the complete vector assembly is performed in vivo by transformation of vector fragments into yeast cells as described previously in the art (Kuijpers et al. 2013) followed by isolation of the complete vectors using commercially available kits.

[0395] Complete vectors comprise the known necessary origin of replication sequences for maintenance of said vectors in the respective organism, the nucleotide sequence to be expressed (selected from: SEQ ID NO:, 9, 10, 11, 12, 13, 14, 15, 16), suitable regulatory elements for expression in the respective organism (at least a promoter and a terminator) and a selection marker allowing the screening of correct transformants and maintenance of the vector in transformed strains. Genomic integration cassettes comprise the nucleotide sequence to be expressed (selected from: SEQ ID NO: 9, 10, 11, 12, 13, 14, 15, 16), suitable regulatory elements for expression in the respective organism (at least a promoter and a terminator), and, optionally, 5′ and 3′ regions with suitable length, such as from 30-3000 bp, which are homologous to the respective genomic integration site to facilitate homologous recombination. In some instances, the genomic integration cassettes are fused and/or co-transformed with suitable selection markers to facilitate screening. In some instances, the suitable selection markers fused with the genomic integration cassette are removed from the final production strain after transformation using methods generally known in the art such as counter-selection in suitable media, use of the Cre-Lox recombinase system or use of CRISPR/Cas methods, such as to create a marker-free strain. In some instances, the nucleotide sequences to be expressed from the vectors and/or genomic integration cassettes are operably linked to sequences encoding for suitable signal peptides to facilitate excretion of the expressed fused protein and/or sequence tags to facilitate the purification of the expressed fused protein after its excretion. In some instances, the operably linked sequences are such as to resulting in the expressed peptides being fused to the N-terminus of the fusion protein. In some instances, the fused sequence comprises recognition sites for native or synthetic peptidases which facilitate cleavage of the signal peptide and/or tag from the fused protein upon or after excretion of the expressed fused protein by the cell.

Example 1.2: General Strain Construction

[0396] Pichia pastoris, Escherichia coli and Aspergillus niger strains are transformed using molecular toolbox techniques known in the art. Selection of correct transformants is performed under known suitable growth conditions for each organism, in suitable known selective growth media such as comprising antibiotics, depending on whether a selection marker is used and its type (dominant/auxotrophic), after allowing for sufficient time for colony growth to be observed. Correct transformants in each case are confirmed by diagnostic PCR or other suitable known molecular toolbox methods such as Southern blotting. In some instances wherein genomic integration cassettes are used for transformation, the strains to be transformed are deficient in non-homologous end-joining repair machinery to facilitate homologous recombination. In some instances wherein a selection marker was used for transformation, the selection marker is removed as described in Example 1, such as to generate a marker-free strain. Correctly transformed strains are preserved in glycerol stocks at −80° C. for further use.

Example 1.3: Production of Myoglobin by Transformed Strains

[0397] Transformed strains obtained in Example 2 are tested for production of extracellular myoglobin. Cell culturing is performed by cultivation in shake-flasks, under conditions conducive to the production of the protein suitable for each host which are generally known, such as temperature, pH and ionic strength of the growth medium, and agitation speed. In some instances, the temperature value ranges from 16-40° C., the pH value ranges from 3-7, the ionic strength value ranges from 100-1000 mM and the agitation speed ranges from 100-300 rpm. The growth medium suitable for cultivation of each host is generally known and comprises carbon and nitrogen sources, as well as additional nutrients such as inorganic salts and vitamins. Cultures are inoculated from frozen stocks of the transformed strains. After 12-24 h of culture, the culture broth is collected and the biomass is removed via centrifugation. Culture supernatant is then tested for the presence of the produced myoglobin using known techniques such as SDS-PAGE electrophoresis by identification of the correct size band or by Western blotting using commercially available antibodies. The results confirm the production of extracellular myoglobin. The produced myoglobin is subsequently purified from the culture supernatant and purified by chromatographic techniques according to standard protocols. The absence of the excretion peptide (cleaved during excretion) is confirmed via mass spectrometry analysis according to standard protein sequencing protocols. The purified myoglobin is stored at −20° C. for later use.

Example 1.4: Production of a Meat Substitute

[0398] Purified myoglobin produced in Example 3 is used for the production of muscle tissue and fat (adipose) tissue according to known procedures. To produce a muscle tissue substitute, purified myoglobin is cross-linked with pea vicilin protein via transglutaminase. A muscle tissue substitute is also produced by forming a heated gel of pea vicilin protein, to which purified myoglobin is added and thoroughly mixed during cooling down of the heated gel to room temperature. A muscle tissue substitute is also produced by co-extrusion of purified myoglobin with pea vicilin protein. A fat tissue substitute is produced by forming a heated gel of pea albumin protein, oil and lecithin following high pressure homogenization, to which the purified myoglobin is added and thoroughly mixed during cooling down of the heated gel to room temperature. A connective tissue substitute is produced using a zein protein source by extrusion according to known procedures. The muscle, fat and connective tissue substitutes are combined in desired ratios in a meat grinder to produce the meat substitute. In some instances, the meat substitute is then cooked.

Example 1.5: Production of Purified Myoglobin by Escherichia coli

[0399] Full length myoglobin genes from woolly mammoth, steppe mammoth, sheep, cow, pig, chicken and tuna (SEQ ID NOs 9, 10, 11, 12, 13, 14, 15, 16) are synthesized and cloned in a modified pET-23a(+) vector, comprising the T7 promoter and terminator and a C-terminal hexa His-tag (Genscript Biotech, Leiden, the Netherlands). Herein, it is understood that SEQ ID NO: 9 codes for only part of a myoglobin, but may nevertheless be synthesized and cloned in said vector. The ampR marker gene originally present in said vector is replaced by the proBA operon from E. coli strain K12, including its original transcription regulatory elements, to facilitate selection without antibiotics. The correctly assembled plasmids are confirmed by PCR and used to transform a proline auxotrophic E. coli protein production strain (E. coli K12 ΔproBA). Transformed strains are incubated overnight in shake-flasks containing minimal medium (10.5 g/L K.sub.2HPO.sub.4, 4.5 g/L KH.sub.2PO.sub.4, 1.0 g/L (NH.sub.4).sub.2SO.sub.4, 0.12 g/L MgSO.sub.4, 0.5 g/l Nacitrate, 2 g/L glucose, and 5.0 mg/L thiamine.Math.HClat 37° C. and 150 rpm (pH 6). 500 μl of the overnight culture is transferred to a 1 L shake-flask containing 500 mL minimal medium and incubated at 37° C. and 150 rpm, until an OD600 of 0.4-1 is reached. IPTG (isopropyl-β-D-thiogalactoside) at a concentration of 100 μM is added to the culture, after which the culture is incubated for 24 h at 16° C. and 150 rpm. The culture is harvested and centrifuged at 3500×g (4° C.) for 15 min. The supernatant is discarded and the pellet is dissolved in 50 mL BugBuster Protein Extraction Reagent (Novagen), containing 1 KU Lysozyme/ml (Sigma-Aldrich), 25 U Benzonase® Nuclease and cOmplete™, EDTA-free Protease Inhibitor Cocktail (Roche). The dissolved pellet is incubated for 30 min at 4° C. in a shaker. The centrifugation step is repeated and the cell-free extract (supernatant) is collected and assayed by SDS-PAGE to confirm the production of myoglobins. The production of woolly mammoth, steppe mammoth, sheep, cow, pig, chicken and tuna myoglobins by the respective transformed strains is confirmed by the presence of protein bands of the correct size.

[0400] For purification, the cell-free extracts containing the soluble fraction of proteins is loaded to a HisTrap FF 1 mL column (Cytiva, MA, USA), coupled with an AKTA start system. The column is equilibrated with 20 mM HEPES, 0.4 M NaCl, and 20 mM imidazole, pH 7.5, 1 mL/min flow rate. The protein is eluted with 20 mM HEPES, 0.4 M NaCl, and 400 mM imidazole, pH 7.5. The fractions containing the myoglobins are pooled, concentrated and confirmed by SDS-PAGE and Western Blotting using an anti-histidine tag antibody (Bio-Rad), which confirm the successful purification of all myoglobins. The purified myoglobins are stored at −20° C. for later use.

Example 1.6: Production of Purified Myoglobin by Aspergillus niger

[0401] Full length myoglobin expression cassettes comprising the genes from woolly mammoth, steppe mammoth, sheep, cow, pig, chicken and tuna (SEQ ID NOs 9, 10, 11, 12, 13, 14, 15, 16), under the control of A. niger glaA promoter and terminator, operably linked to the excretion signal sequence of pectinmethylesterase of A. niger to facilitate myoglobin excretion, are synthesized. Herein, it is understood that SEQ ID NO: 9 codes for only part of a myoglobin, but may nevertheless be comprised in said expression cassette Genomic integration cassettes are assembled by fusion PCR of myoglobin expression cassettes with the orotidine 5′-phosphate decarboxylase gene sequence (pyrG) from Aspergillus oryzae, as well as 1000 bp at the 5′ and 3′ ends which are homologous to the native upstream and downstream sequences of the A. niger pyrG gene.

[0402] The genomic integration cassettes are transformed to protoplasts of A. niger CBS 120.49 ΔkusA ΔpyrG. Prior to transformation, protoplasts are prepared by overnight shake-flask cultivation in complete medium (2% (mass/vol) glucose, 6 g/L NaNO.sub.3, 1.5 g g/L KH.sub.2PO.sub.4, 0.5 g/L KCl, 0.5 g/L MgSO.sub.4*7H.sub.2O, 0.2% (mass/vol) tryptone, 0.1% (mass/vol) yeast extract, 0.1% (mass/vol) casamino acids, 0.05% (mass/vol) yeast RNA, and trace elements according to Vishniac (1957)) at 30° C. and 250 rpm. Mycelia are harvested by filtration and dissolved in PS buffer (0.2 M sodium phosphate buffer, 0.8 M L-sorbitol, pH 6), to which 0.5 g VinoTaste® Pro lysing enzyme per g of mycelia is added, followed by incubation at 30° C. and 100 rpm. Undigested mycelia are removed via filtration and the protoplasts are collected by mild centrifugation (1500×g, 3° C.), washed with SC solution (182.2 g L.sup.−1 sorbitol, 7.35 g L.sup.−1 CaCl.sub.2)*2H.sub.2O) and resuspended in the same solution to a concentration of 108 protoplasts/mL. Fresh protoplasts are transformed by mixing 200 μL of protoplast suspension with 5 μg of genomic integration cassette in a mixture additionally containing 20 μL of 0.4 M ATA (aurintricarboxylic acid ammonium salt) and 100 μL of 20% PEG-4000, followed by incubation for 10 min. Then, 5 mL of 1.2 M sorbitol solution are added and the mixture is incubated for another 10 min. The transformed protoplasts are collected by mild centrifugation and resuspended in 1 mL of the same sorbitol solution. Transformed protoplasts are plated in minimal medium agar plates (1.5% (mass/vol) agar, 2% (mass/vol) glucose, 6 g/L NaNO.sub.3, 1.5 g/L KH.sub.2PO.sub.4, 0.5 g/L KCl, 0.5 g/L MgSO.sub.4*7H.sub.2O, and trace elements according to Vishniac (1957)) and incubated at 30° C. for 4 days (pH 5). DNA from selected transformants is extracted using standard phenol/chloroform extraction. Correct integration of genomic integration cassettes comprising the myoglobin genes from woolly mammoth, steppe mammoth, sheep, cow, pig, chicken and tuna are confirmed by Southern blotting.

[0403] Spores of correct transformants are obtained by incubation on complete medium agar plates for four days at 30° C. and harvested with 10 mL N-(2-acetamido)-2-aminoethanesulfonic acid (ACES) buffer. 2×10.sup.8 spores are used to inoculate shake-flask cultures containing 400 mL minimal medium and incubated overnight at 30° C., 250 rpm. Mycelia are transferred to fresh shake-flask cultures containing 400 mL minimal medium and at 30° C., 250 rpm for 24 h. The cells are removed by culture harvesting followed by centrifugation at 3200×g at 4° C. for 10 min. The supernatant is assayed by SDS-PAGE to confirm the production of myoglobins. The expression of woolly mammoth, steppe mammoth, sheep, cow, pig, chicken and tuna myoglobins by the respective transformed strains is confirmed by the presence of protein bands of the correct size.

[0404] For purification, culture supernatants containing the soluble fraction of proteins are loaded to a HiLoad 16/600 Superdex 75 μg column (10 mm×300 mm) (Cytiva, MA, USA). The column is equilibrated with 0.15 M ammonium acetate, pH 6.0, 0.75 mL/min flow rate. The fractions containing the myoglobins are pooled, concentrated and confirmed by SDS-PAGE and LC-MS according to standard protocols. The results confirm the presence of the purified myoglobins without the excretion signal peptide.

Example 1.7. Production of Purified Myoglobin by Pichia pastoris

[0405] Full length myoglobin expression cassettes comprising the genes from woolly mammoth, steppe mammoth, sheep, cow, pig, chicken and tuna (SEQ ID NOs 9, 10, 11, 12, 13, 14, 15, 16), under the control of P. pastoris native pgk1 promoter and terminator, are synthesized. Genomic integration cassettes are assembled by fusion PCR of myoglobin expression cassettes with the gene encoding the native P. pastoris histidine biosynthesis trifunctional protein gene (his4) under control of its own promoter and terminator, as well as 1000 bp at the 5′ and 3′ ends which are homologous to the native upstream and downstream sequences of the P. pastoris his4 locus. The genomic integration cassettes are transformed to the antibiotics-resistance marker free, histidine auxotrophic, P. pastoris strain Bg12 (BioGrammatics Inc. Carlsbad, CA), using the lithium-acetate transformation protocol as previously described in Gietz and Woods (2002) (Gietz R D et al., (2002), Methods Enzymol., 350: 87-96). Correct transformants are obtained by incubation on 2% w/v agar plates containing synthetic medium (without histidine to facilitate selection) as previously described in Verdyun et al. (1992) (Verduyn C., et al (1992), Yeast, 8: 501-517): 5 g/L (NH.sub.4).sub.2SO.sub.4, 3 g/L KH.sub.2PO.sub.4, 0.5 g/L MgSO.sub.4.Math.7H.sub.2O, 4.5 mg/L ZnSO.sub.4.Math.7H.sub.2O, 0.3 mg/L CoCl.sub.2.Math.6H.sub.2O, 1 mg/L MnCl.sub.2.Math.4H.sub.2O, 0.3 mg/L CuSO.sub.4.Math.5H.sub.2O, 4.5 mg/L CaCl.sub.2).Math.H.sub.2O, 3 mg/L FeSO.sub.4.Math.7H.sub.2O, 0.4 mg/L NaMoO.sub.4.Math.2H.sub.2O, 1 mg/L H.sub.3BO.sub.3, 0.1 mg/L KI, 0.05 g/L biotin, 1 mg/L calcium pantothenate, 1 mg/L nicotinic acid, 25 mg/L inositol, 1 mg/L thiamine.Math.HCl, 1 mg/L pyridoxine.Math.HCl, 0.2 mg/L para-aminobenzoic acid and 2% w/v glucose as carbon source (pH 5). Plates are incubated at 30° C. for 4 days. Correct integration of genomic integration cassettes comprising the myoglobin genes from woolly mammoth, steppe mammoth, sheep, cow, pig, chicken and tuna is confirmed by colony PCR, following DNA preparation using a Yeast Protein Kit (ZymoResearch, Irvine, CA), according to manufacturer's protocol. Glycerol stocks of correct transformants are prepared and stored at −80° C.

[0406] For myoglobin production, frozen glycerol stocks are used to inoculate 1 L-pre-culture shake-flasks containing 500 mL of synthetic medium and incubated overnight at 30° C. and 250 rpm. The pre-cultures are used to inoculate subsequent shake-flask cultures to a starting OD660 of 0.2. Cultures are incubated at 30° C. and 250 rpm for 24 h. The cultures are harvested and centrifuged at 3500×g (4° C.) for 15 min. The supernatant is discarded, the cell pellet is resuspended in ice-cold demineralized water and the centrifugation step is repeated. The supernatant is discarded and the cells are lysed using a Yeast Protein Kit (ZymoResearch, Irvine, CA), according to manufacturer's protocol, together with mechanical disruption. The mixture containing the cellular debris and the soluble proteins is centrifuged at 3500×g (4° C.) for 15 min. The supernatant (cell-free extract) containing the soluble proteins is collected and assayed by SDS-PAGE to confirm the production of myoglobins. The production of woolly mammoth, steppe mammoth, sheep, cow, pig, chicken and tuna myoglobins by the respective transformed strains is confirmed by the presence of protein bands of the correct size.

[0407] For purification, cell-free extracts containing the soluble fraction of proteins are loaded to a HiLoad 16/600 Superdex 75 μg column (10 mm×300 mm) (Cytiva, MA, USA). The column is equilibrated with 0.15 M ammonium acetate, pH 6.0, 0.75 mL/min flow rate. The fractions containing the myoglobins are pooled, concentrated and confirmed by SDS-PAGE and LC-MS according to standard protocols. The results confirm the presence of the purified myoglobins.

Example 2: production Method B

[0408] Unless explicitly mentioned otherwise, “mammoth” and “mammoth myoglobin” refer to “steppe mammoth” and “steppe mammoth myoglobin” in Example 2.

Example 2.1. Materials and Methods

Sequence Analysis

[0409] The sequence coding for myoglobin from Bos taurus, Gallus gallus, Thunnus orientalis, Ovis aries and Sus scrofa were obtained from Uniprot (accession numbers: P02192, P02197, P68190, P02190, P02189, respectively). The sequence coding for myoglobin from the steppe mammoth (Mammuthus Trogontherii), which was unknown at the time this application was filed, was obtained by the inventors after DNA extraction from a molar sample from the so-called Adycha specimen, Illumina DNA sequencing, merging the reads and mapping them against the African savannah elephant (Loxodonta africana) genome (van der Valk et al. 2021).

[0410] Multiple sequence alignments were performed with Clustal Omega (Sievers et al. 2011) and visualized using Jalview 2.11.1.4 (Waterhouse et al. 2009).

Protein Modelling

[0411] The structure of wildtype deoxymyoglobin from Sus scrofa was retrieved from the RCSB Protein Data Bank (PDB accession number: 1MWD, chain A). The structure of the steppe mammoth myoglobin was modelled using the automated protein structure homology-modelling server SWISS-MODEL (Waterhouse et al. 2018), using the crystal structure of myoglobin from Elephas maximus (asian elephant) as template (PDB accession number: 1EMY). Net surface charge (Z.sub.Mb) was calculated as the sum of the charge of all ionizable groups at pH 6.5 using published, site-specific ionization constants (Mirceta et al. 2013). All protein structures were visualized and figures generated using DeepView v4.1 (Guex and Peitsch 1997).

Construction of Expression Plasmids

[0412] Myoglobin coding sequences were codon optimized for expression in Pichia pastoris (Komagataella phaffii) (Love et al. 2016) (SEQ ID NO: 19-24). The optimized sequences, preceded by the coding sequence of the Saccharomyces cerevisiae mating factor alpha (SEQ ID NO: 25-30) were chemically synthetized by GenScript. Gene fragments were cloned into the pBDIPp5 vector, downstream of the AOX1 promoter and upstream of the AOX1 terminator, the HIS4 selection marker and the AOX1 3′ fragment. Vectors were amplified in E. coli (DH10B), purified with the Plasmid kit (Quiagen) and verified by Sanger sequencing. Expression cassettes were generated from the resulting vectors by restriction of the plasmids with BglII (New England Biolabs), which cuts upstream from the AOX1 promoter and downstream from the AOX1 3′ fragment, and purified with the QIAquick Gel Extraction Kit (QUiagen).

Strains Engineering

[0413] The Pichia pastoris (Komagataella phaffii) strain GS115 (his4) was obtained from Life Technologies. Cell transformation was performed using the electroporation method essentially as previously described (Cregg 2007). Briefly, cells of the GS115 strain were grown in YPD (1% yeast extract, 2% peptone and 2% D-glucose) medium. Cells in the exponentially growing phase were incubated for 30 min in YPD medium with 200 mM HEPES buffer (pH 8.0) and 25 mM dithiothreitol. Competent cells were then washed with ice-cold 1M sorbitol, and transferred to a sterile electroporation cuvette (Bio-Rad). Cells were then electroporated with 1-5 μg of linear expression cassette (see 3.3) using a Gene-Pulser (Bio-Rad) electroporator, and resuspended in 1 mL of YPD medium containing 1M sorbitol before transfer to a sterile 1.5-mL eppendorf tube. Cells were incubated at 28° C. without agitation for 3 h, before plating onto agar plates containing solid MGY medium (Minimal Glycerol Medium: 1.34% Yeast Nitrogen Base with ammonium sulfate without amino acids, 2% D-glucose, 4 10-5% biotin with 2% agar). Plates were incubated for up to 4 days at 28° C.

[0414] Transformants able to grow in the absence of histidine were screened for their ability to express Mb. Cells were grown in 50 mL Falcon tubes in BMGY medium (1% yeast extract, 2% peptone, 100 mm Potassium Phosphate buffer (pH6), 1.34% Yeast Nitrogen Base with ammonium sulfate without amino acids, 4 10-5% biotin, 1% glycerol). 1% methanol was added to exponentially growing cells, to induce Mb expression. Samples were collected 24 h, 48 h, 72 h and 96 h after the start of the methanol induction, and analyzed by SDS-PAGE to assess Mb levels (see below).

[0415] The best producing strains were selected for further experiments, and were cryopreserved as a master cell bank at −80° C. prior to use. Results shown here were obtained using the PAL-02-02 (mammoth Mb) and PAL-02-03 (cattle Mb) strains.

Recombinant Protein Production and Purification

[0416] Cells from the master cell bank were plated on a YPD agar (1% yeast extract, 2% peptone and 2% D-glucose, 2% agar) plate and incubated for 2 days at 28° C. A seed culture was then prepared in a baffled flask containing 100 mL of BMGY medium, and incubated for 24 h at 28° C. in an orbital shaker incubator. The seed culture was then used to inoculate a 3 L spherical glass flask or a glass-vessel fermenter (Sartorius) containing 900 ml of BMGY medium with 0.03% FoamAway™ Irradiated AOF (ThermoFisher). The propagation phase was conducted for about 24 h, until all the glycerol was consumed. The temperature was then lowered to 26° C., and methanol 1% was added every 12 h to induce Mb expression. Dissolved oxygen was maintained above 20% during the whole fermentation, while the pH was kept at 6 during the propagation phase and 5 during the induction phase.

[0417] After 96 h of methanol induction, cells were removed by centrifugation at 4,000 rpm at 4° C. Remaining cells were removed by microfiltration using nitrocellulose filters with a pore diameter of 0.45 μm (Millipore). The cell-free supernatants were stored frozen at −20° C. Where needed for the assays, cell-free supernatants were further concentrated by using disposable ultrafiltration centrifuge devices, with a polyethersulfone membrane having a molecular-weight cutoff of 10 kD (Thermo Scientific Pierce Protein Concentrators).

[0418] The presence of recombinant DNA in the cell-free supernatant was tested by PCR, using oligonucleotides (Sigma) designed to be complementary to the sequence encoding the signal peptide. A DNA fragment amplified from the genomic DNA of the PAL-02-02 strain (mammoth Mb) was used as positive control, next to 1 μl of concentrated cell-free supernatant containing the mammoth or cattle Mb. PCR reactions were conducted using the OneTaq® Quick-Load® 2× Master Mix with Standard Buffer (New England Biolabs) according to the manufacturer's instructions. PCR products were vizualized after migration at 60V for 60 min in a 2% agarose TAE gel with ethidium bromide. The the Quick-Load® Purple 1 kb Plus DNA Ladder (New England Biolabs) was used to control the size of the amplified fragment.

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

[0419] For protein electrophoresis, 20 μl of concentrated cell-free supernatant were mixed with an equal volume of 2× protein loading buffer (100 mM Tris (pH6,8); 4 mM EDTA, 4% SDS, 20% glycerol; 0.02% bromophenol blue, 4% β-mercaptoethanol). Samples were incubated at 95° C. for 5 minutes, and the appropriate volume loaded in a 15% polyacrylamide gel on a vertical mini-PROTEAN gel apparatus (Bio-Rad) at 200 V for 40 minutes. The molecular weight marker (PageRuler™ Prestained Protein Ladder) was purchased from Thermofisher. After electrophoresis, proteins in the gel were stained with Coomassie Brilliant Blue G-250 (Bio-Rad).

Mass Spectrometry

[0420] For MS analysis, the SDS-PAGE gel band corresponding to Mb was excised from the Coomassie-stained gel using a scalpel knife. The sample was rinsed with sterile water and stored at −20° C. until processed for MS analysis at the VIB Proteomics Core (Gent, Belgium). Peptides were generated by digestion with 1 μg trypsin (Promega) overnight at 37° C., and stored at −20° C. until LC-MS/MS analysis. Peptides were re-dissolved in loading solvent and injected for LC-MS/MS analysis using a Q Exactive HF mass spectrometer (Thermo Fisher). Data analysis was performed with MaxQuant algorithm (version 2.0.1.0) with default search settings including a false discovery rate set at 1% on peptide-spectrum match (PSM), peptide and protein level. All raw spectral data files were searched together against the theoretical protein sequence of mammoth Mb and the following reference proteomes in the Uniprot database: Komagataella phaffii (database release version of 2021_11, containing 5,073 protein sequences), Bos taurus (taxid 9913, database release version of 2021_11, containing 37,513 protein sequences), and Sus scrofa (taxid 9823, database release version of 2021_11, containing 49,792 protein sequences).

Plant-Based Meat Analogues

[0421] Plant-based burgers were prepared by mixing 25% texturized soy proteins, 15% sunflower oil, 1.5% NaCl, 1% methylcellulose and 57.5% water. Recombinant myoglobin or commercially available myoglobin (purified from equine muscle, Sigma) were added at 0.5% or 1% final concentration, similar to the myoglobin content of white and red meat, respectively. Recombinant myoglobin preparations were obtained by lyophilization of the cell-free supernatant. For the burgers containing 1% Mb, 13.8 g of additional water were added per 100 g of burger. Commercially available plant-based burgers containing soy Leghemoglobin (Impossible Food) were included for comparison in some assays.

Spectrometry and Color Analyses

[0422] Absorbance spectra were recorded with a Synergy microplate reader (BioTek). Absorbance measurements for the determination of myoglobin auto-oxidation rate were performed in quartz cuvettes using an Ultrospec III spectrophotometer (Pharmacia). Color measurements were performed using a portable Miniscan EZ 4500 L 45°/0° (Hunterlab, Murnau, Germany) with 8 mm viewing area size, illuminant D65 and 10° standard observer to register the L*, a*and b* values (based on the CIELAB color space). The difference in color over time (ΔE) was calculated according the CIE76 formula:

ΔE-value (−)

ΔE=√{square root over ((ΔL*).sup.2+(Δa*).sup.2+(Δb*).sup.2)}

Aromatic Analyses

[0423] Aromas from plant-based meat alternatives containing different concentrations of recombinant Mb were analyzed using gas chromatography-mass spectrometry (GC-MS) with headspace solid-phase microextraction (HS-SPME). Samples were analyzed raw or after grilling. The headspace volatiles were extracted by Divinylbenzene-Carboxen-Polydimethylsiloxane (DVB/CAR/PDMS) coated fibers, and separated by an HP-1 ms column, before identification by mass spectrometry. For data analysis, the area under the peaks of the different aromatic compounds was evaluated by one-way ANOVA (analysis of variance), followed by Tukey's HSD (Honestly Significant Difference) posthoc test when statistically significant differences were found. Multivariate statistical analysis was performed using PCA (Principle Component Analysis).

Iron Bioavailability and Bioaccessibility

[0424] Assays were performed by ProDigest (Gent, Belgium). Caco-2 cells (HTB-37; American Type Culture Collection) were seeded at 5×105 cells in 12-wells coated with 0.1% gelatin. Cells were grown for 14 days in complete medium (Dulbecco's Modified Eagle Medium (DMEM) supplemented with 20% heat-inactivated fetal bovine serum (FBS), 10 mM HEPES and 1× antibiotic-antimycotic) with 3 medium changes/week. 24 h prior to stimulation, cells were washed once with Minimum Essential Medium (MEM) supplemented with 10 mM HEPES, 2 mM L-Glutamine, 1× antibiotic-antimycotic, 11 μM hydrocortisone, 0.87 μM insulin, 0.02 μM sodium selenite, 0.05 μM 3,3′,5-Triiodo-L-thyronine sodium salt, 20 μg/L epidermal growth factor and further incubated in this medium for 24 h. Then, cells were incubated with bovine Mb (Tebu-bio N.V.) at three concentrations (0.5, 1 and 2 mg/mL) or with supplemented MEM as negative control (indicated as CM). After 24 h of incubation at 37° C., cells were washed twice with ice-cold PBS and lysed with CelLytic™ (Sigma Aldrich). Human ferritin levels were determined using a human ferritin ELISA kit (ThermoScientific) according to the manufacturer's instructions. Finally, protein concentrations were determined using Pierce™ BCA Protein Assay kit (ThermoFisher Scientific) following the microplate procedure instructions. All assays were performed in triplicate.

In Vitro Toxicity Assay

[0425] Assays were performed by ProDigest (Gent, Belgium). Caco-2 cells were seeded in 24-well plates coated with 0.1% gelatin an cultivated as for the ferritin assay. On the day of the toxicity test, cells were incubated with recombinant mammoth or cattle Mb at 0.5, 1.0 or 2.0 mg/ml. Cytotoxicity assays were performed after 24 h of incubation with Mb at 37° C. To assess possible toxicity induced by the products on Caco-2 cells, a lactate dehydrogenase (LDH) cytotoxicity assay (Merck Life Science B.V.) was performed on the supernatants, according to the manufacturers' instructions. All assays were performed in triplicate. To assess differences between the complete medium control (CM) and the products, an ordinary one-way ANOVA with Dunnett's multiple comparisons test was performed for each timepoint separately. (*) represents statistically significant differences between CM and products. (*)=p<0.05; (**)=p<0.01; (***)=p<0.001 and (****)=p<0.0001. All statistics were performed using GraphPad Prism version 9.1.2 for Windows (Graph Pad Software, San Diego, CA, USA).

Bacterial Reverse Mutation Assay (AMES Test)

[0426] The genotoxicity assay was performed according to the OECD Guideline for chemical testing no. 471. The AMES FT Mutagenicity Test Kit (Moltox, Trinova) was used following the manufacturer's instructions. All assays were performed in triplicate. The mean revertant counts for each strain treated with the vehicle were within the expected range based on data provided by the manufacturer. Mutation rates were calculated as the ratio between the number of revertants observed with the test compound and with the vehicle alone.

Example 2.2. Structural Characteristics of Myoglobin

[0427] Myoglobin (Mb) is a relatively small globular protein of about 17 kD, found in heart and skeletal muscles. It carries a single heme group capable of reversible oxygen binding (FIG. 1), allowing myoglobin to transport oxygen from the cell surface to mitochondria. Mb is also the primary pigment responsible for meat color. In oxygenated Mb (oxymyoglobin), the iron atom of the heme group is coordinated by four nitrogen atoms of the porphyrin ring, the so-called “proximal” histidine (His94) in the Mb chain, and an oxygen molecule. This can be further stabilized by hydrogen bonding between the oxygen molecule and another histidine in the heme binding pocket of Mb, the “distal” histidine (His65). Under this form, Mb has a typical bright red color. In the absence of oxygen, Mb takes a darker red color (deoxymyoglobin). When the heme iron is oxidized from the ferrous (Fe(II)) to the ferric (Fe(III)) state, it is unable to bind oxygen and Mb displays a brown color (metmyoglobin), as seen in cooked meat. Heme iron oxidation also reduces the affinity of Mb for heme, leading to increased heme loss and subsequent unfolding of the protein.

[0428] The amino acid sequence of Mb has been strongly conserved during evolution, and shows relatively little variation between species (FIG. 2). In Proboscideans, Mb presents an atypical phenylalanine in position 30 (Phe30). Remarkably, the Mb sequence from a steppe mammoth (Mammuthus trongotherii), which we generated from the so-called Adycha specimen, dating between 1.2 and 1.0 Ma, is also included in FIG. 2.

[0429] In addition to the characteristic Phe30 substitution (FIG. 2), we found that Mb from steppe mammoth displayed a higher positive surface charge (net surface charge (Z.sub.Mb) at pH 6.5=2.67) than Mb from e.g. the asian elephant (Z.sub.Mb at pH 6.5=2.11), due to the presence of a histidine residue instead of a glutamine at position 92 (His92) (FIG. 2, 3). This is reminiscent of adaptation of Mb to deep-sea diving in cetaceans, where increased positive surface charge led to decreased attractive interactions between Mb molecules at a contact distance, increasing Mb stability and preventing its aggregation. Overall, Mb from the steppe mammoth remarkably presents several characteristics that make it particularly attractive. Here, we evaluate the production and characteristics of recombinant Mb from the steppe mammoth, next to other extant species.

Example 2.3. Extracellular Production of Myoglobin in Pichia pastoris

[0430] The sequences coding for Mb from the steppe mammoth, pig, chicken, cattle, pork and tuna were cloned downstream of the methanol-inducible AOX1 promoter of Pichia pastoris, and upstream of the AOX1 terminator, a histidine prototrophy selection marker and the so-called 3′ fragment of the AOX1 gene. Myoglobin is naturally found in the cytoplasm of muscle cells. To facilitate recombinant protein purification, we fused a sequence encoding a signal peptide in frame with the myoglobin coding sequence, in order to target nascent Mb proteins to the secretory pathway for excretion outside the cell. These constructs were used to transform histidine-auxotroph Pichia pastoris cells, and transformants were selected for their ability to grow in the absence of histidine. For each construct, up to ten transformants were tested for their ability to produce extracellular animal myoglobin after methanol induction in microplates. The best producing clones were further tested in flasks. Methanol induction led to the cell-free supernatant displaying a dark red color, especially after 10-fold concentration by ultrafiltration, as expected for samples containing a heme protein (FIG. 4A). The presence of a methanol-inducible protein of the expected molecular weight, about 17 kD, was also confirmed after protein electrophoresis and staining with Coomassie blue (FIG. 4B). Recombinant Mb accounted for 75-82% of the total proteins in the concentrated cell-free supernatant, while the yield for Mb production varied between 0.420 mg and 1.93 g per liter of supernatant.

[0431] Since myoglobin is produced extracellularly, yeast cells do not need to be lyzed during the purification process. These cells are removed from the fermentation broth by centrifugation, followed by microfiltration (0.45 μm pores). The final product is therefore not expected to contain any recombinant genetic material. To verify this, we designed a PCR test based on the amplification of a short fragment (130 bp) of the recombinant gene, corresponding to the sequence encoding the signal peptide, from as little as 1 μg of DNA (FIG. 5). Using this test, we failed to detect any recombinant DNA in the concentrated cell-free supernatant (FIG. 5).

[0432] In order to confirm identity of the recombinant Mb, and ensure that the signal peptide was correctly processed and removed during protein secretion, we analyzed the mammoth Mb gel band by liquid chromatography coupled to mass spectrometry (LC-MS/MS) shotgun measurement. We recovered peptides covering of 90.3% of the full protein sequence and observed complete removal of the signal peptide and the first methionine, as expected (FIG. 6).

Example 2.4. Color and Color Stability of Recombinant Myoglobin

[0433] As discussed above, Mb plays a central role in meat color. In parallel with the mass spectrometry analysis, we therefore measured absorbance of the concentrated cell-free supernatants in the UV and visible range, and compared it with the absorbance of commercial myoglobin purified from equine muscle. In all cases, an absorbance peak (Soret band) characteristic of the presence of heme was observed (Tang et al. 2004). The Soret band was detected at 410 nm for the cattle and horse Mb, while it was slightly red-shifted to 415 nm for the mammoth Mb (FIG. 7A). Based on the absorbance ratios at the wavelength maxima representative for metmyoglobin, deoxymyoglobin, and oxymyoglobin, respectively (Tang et al. 2004), we observed that recombinant myoglobin in the concentrated cell-free supernatant is mainly found in the reduced form: 47.3% oxymoyglobin and 10.0% deoxymyoglobin for the mammoth Mb, 48.3% oxymoyglobin and 9.9% deoxymyoglobin for the cattle Mb. Of note, the Soret peak of mammoth Mb was consistently observed at a slightly higher wavelength than for the cattle and horse Mb (415 nm versus 410 nm, respectively). This is consistent with previous observations made for elephant Mb (Tada et al. 1998). In order to assess the auto-oxidation behavior of Mb, we recorded absorbance spectra every two hours during a 24-hour incubation at pH 5.6 and 25° C. (FIG. 7B). We then examined the ratio between absorbance at 580 nm (peak value for oxymyoglobin, carrying Fe(II)) and at 505 nm (peak value for metmyoglobin, carrying Fe(III)). At the start of the incubation, we observed that this ratio was significantly higher for the recombinant mammoth and cattle Mb than for commercially available Mb purified from equine muscle (FIG. 7B, insert), indicating that a larger fraction of recombinant Mb is present in the reduced form. This difference with horse Mb was maintained over time at acidic pH (FIG. 7B, insert). Over the course of 24 hours, the absorbance ratio decreased by 26.0% for the mammoth Mb and 38.3% for the cattle Mb, suggesting that the former shows a greater resistance to auto-oxidation.

[0434] Meat color is affected by multiple parameters, including Mb concentration, moisture and fat content. For traditional meat, which is typically purchased raw, the red color imparted by oxymyoglobin plays a key role in consumers purchase decision. We therefore tested the effect of recombinant Mb on meat analogues in terms of color. Addition of cattle or mammoth Mb to plant-based burgers (FIG. 8A-A) caused a decrease in lightness (L*-value), and an increase in redness (a*-value) and yellowness (b*-value) (FIG. 8B).

[0435] Next, we assessed color stability of meat analogues containing various amounts of myoglobin, which were stored in a cold room and exposed to constant light for 5 days. A commercial plant-based burger containing a soy heme protein was included for comparison. While we observed significant color change for the burger prepared without Mb, the addition of Mb was associated with a greater color stability (FIG. 9A-B).

Example 2.5. Aromatic Analysis

[0436] Besides its role in meat color, myoglobin is generally assumed to contribute to the “bloody” or “metallic” smell of raw meat. During meat cooking, aroma formation occurs essentially by lipid oxidation and the Maillard reaction. The later takes place between amino groups in proteins and carbonyl groups from reduced sugars and/or reaction products of lipid oxidation. Heme iron can influence these reactions and/or their kinetics (van Ba et al. 2012). Yet, to our knowledge there is no conclusive data in the literature about the influence of myoglobin on aroma formation in meat. Using lab-made burgers as described above, we analyzed volatile compounds from raw and cooked plant-based burger containing recombinant Mb by gas chromatography coupled with mass spectrometry.

[0437] We found that the addition of Mb to a plant-based burger significantly increased the number of volatile compounds, both in the raw state and after grilling (FIG. 10A). Noticeably, the number of volatiles produced was higher in both case than with a commercial plant-based burger containing soy leghemoglobin (FIG. 10A), i.e. the Impossible Burger. Using Principal Component Analysis (PCA) on the CG-MS data, we observed a clear separation between lab-based burgers and commercially available plant-based burger containing soy leghemoglobin on the one hand, and between raw and baked products on the other hand (FIG. 10B).

[0438] When analyzing the volatile compounds from the grilled lab-made plant-based burgers, we found that addition of Mb was associated with the presence of oxidized lipids, lipid oxidation products and pyrazines. In particular, the presence of steppe mammoth or cattle Mb led to increased amounts of compounds known to confer a “roasted” taste to grilled meat (van Ba et al. 2012) (FIG. 12). The addition of mammoth Mb was consistently associated with a higher amount of these compounds than what was observed with the cattle Mb. This suggests that, without being bound to this theory, when used as an ingredient for plant-based meat analogues, lower amounts of mammoth Mb could be added to obtain the same aromatic properties than when using cattle Mb. Additionally, the mammoth Mb and cattle Mb might provide a different sensory experience.

Example 2.6. Nutritional Analysis

[0439] In order to assess the bioavailability of iron from Mb, we turned to the epithelial-like intestinal cell line Caco-2, which is commonly used to study human intestinal iron uptake. We measured the formation of intracellular ferritin as a readout of iron uptake by a living Caco-2 cell monolayer, and therefore an indicator of iron bioavailability (Glahn et al. 1998). We observed that increasing doses of Mb led to an increase in iron uptake (FIG. 12), indicating that heme-iron carried by Mb is bio-available to human intestinal cells.

Example 2.7. Safety Testing

Cytotoxicity

[0440] In order to assess the potential toxicity of recombinant Mb, differentiated Caco-2 cells were treated with the mammoth or cattle Mb, with purified bovine Mb or with complete medium (CM) as a control. To test for potential cytotoxic effects, we then examined the levels of lactate dehydrogenase (LDH) in the culture supernatant. LDH is an oxidoreductase present in all cells, which catalyzes the interconversion of pyruvate to lactate with concomitant interconversion of NADH to NAD+. Upon cell membrane damage following apoptosis or necrosis, LDH is released in the supernatants and its concentration can be determined by a colorimetric assay based on the reduction of NAD+ to NADH by LDH (Decker and Lohmann-Matthes 1988). We did not observe an increase in cytotoxicity upon incubation with mammoth Mb at any of the concentrations tested (FIG. 13). In contrast, the cattle Mb showed significantly increased toxicity compared to the medium alone (FIG. 13). At the high concentrations tested here, the mammoth Mb therefore appears to have a better safety profile than cattle Mb.

Genotoxicity

[0441] In order to determine the mutagenic potential of recombinant myoglobin, we used a bacterial reverse mutagenesis test (AMES test) and four histidine-requiring strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537) and one tryptophan-requiring strain of Escherichia coli (WP2 uvrA) (Ames et al. 1975). Bacteria were exposed to mammoth or cattle myoglobin at levels of 0.8, 2.5, 8.0, 25.0, 80.0 or 250 μg/ml, either with or without exogenous metabolic activation (S9 mix) (Table 1). The positive control substances caused the expected increase in number of revertants, both the absence and the presence of S9, confirming the sensitivity of the test and the activity of the S9 mix. We did not observe any significant mutagenic activity for the cattle or mammoth Mb at any of the concentrations tested.

TABLE-US-00004 TABLE 1 Number of revertants and mutation factor after exposure to mammoth myoglobin. The mean number of revertants observed for a total of 48 wells per condition is indicated in black. The corresponding mutation factor (ratio of the revertant counts in presence of a compound versus the vehicle alone) is indicated in grey. All assays were performed in triplicates. Conditions where significant mutagenesis was observed are highlighted in bold. Compounds used as positive controls were a2-nitrofluorene 2 μg/ml, baminoanthracene 4 μg/ml, cN4-aminocytidine 100 μg/ml. AMES test Strain TA98 TA100 TA1535 Test compound −S9 +S9 −S9 +S9 −S9 +S9 Vehicle 1.33 ± 1.53 1.67 ± 2.08 8.00 ± 2.00 6.00 ± 5.20 2.33 ± 1.53 3.00 ± 1.0  Mb 0.003 μg/μl 0.00 ± 0.00 1.00 ± 0.00 10.0 ± 2.65 7.00 ± 2.65 2.33 ± 0.58 2.33 ± 0.58 0.00 ± 0.00 0.60 ± 0.00 1.25 ± 0.33 1.17 ± 0.44  1.0 ± 0.25 0.78 ± 0.19 Mb 0.003 μg/μl 2.33 ± 2.52 1.33 ± 1.58 9.33 ± 2.52 4.67 ± 2.08 1.00 ± 1.00 2.67 ± 0.58 1.75 ± 1.89 0.80 ± 0.35 1.17 ± 0.31 0.78 ± 0.69 0.43 ± 0.43 0.89 ± 0.19 Mb 0.003 μg/μl 1.33 ± 1.53 1.33 ± 2.31 12.33 ± 7.77  9.00 ± 1.73 2.37 ± 1.15 2.33 ± 1.15 1.00 ± 1.15 0.80 ± 1.39 1.54 ± 0.97 1.50 ± 0.58 1.14 ± 0.49 0.78 ± 0.38 Mb 0.003 μg/μl 0.33 ± 0.58 1.00 ± 1.00 14.67 ± 5.51  7.00 ± 2.00 1.00 ± 1.00 3.00 ± 1.00 0.25 ± 0.43 0.60 ± 0.60 1.83 ± 0.69 1.17 ± 0.67 0.43 ± 0.43 1.00 ± 0.33 Positive 37.33 ± 2.08a 48.00 ± 0.00b 47.67 ± 0.58c 44.00 ± 5.20b 48.00 ± 0.00c 21.33 ± 1.50b control 28.0 ± 1.56 28.80 ± 0.00  5.96 ± 0.07 7.33 ± 1.73 20.57 ± 0.00  7.11 ± 0.51