Involuntary graphene intake with food and medicine

Abstract

Graphene is found in charred roasted meat and also in plant charcoal, which is present in the infant's gripe water. Graphene as graphene oxide (GO) is produced on charring the surface of meat on a barbecue forming nitrogen doped GO originating from the pyrolysis of protein in air. The unintentional intake of such nano carbon stained cooked meat is a traditional delicacy. The presence of graphene and nano carbon particles in plant charcoal, used in the branded formulation of gripe water applied for stomach ailments in infants as a medicine, strongly refutes the toxicity of such nano carbons in humans. The isolation of graphene and nano particulates from both the sources is described here. These are characterized by elemental analysis, Raman spectroscopy, PXRD and by FESEM, TEM and SADP image analysis. The intake of nano carbon contaminated roasted food since the discovery of fire possibly trails mutation to evolve resistance in humans. This work suggests that graphene and other nano carbon particles produced by the pyrolysis of bio-products in air are non-toxic to humans.

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1. Introduction

The beneficial or deleterious effect of nano-materials present in our ecosystem has been debated. Such issues have gained importance because of the recent rise in nano-material research.^1–6 One aspect of nano material–human interaction may be exterior as external application^7,8 that may be readily avoided if found harmful. However, interior involvement of nano-materials may have serious consequences, which can only be traced by in vitro or by in vivo studies.^1,5,6 Current research is aimed at developing biocompatible nano-materials to avoid possible adverse effects.^9,10 However, the question still remains as how long these are in trial use to justify them as absolutely safe by current health protocols. Such application virtually requires an in depth investigation similar to what is required for a new drug to confirm its safety. Therefore, the introduction of any new nano-material for the benefit of humans requires several queries to be answered that may involve a long waiting time for systematic procedural tests to conclude. Further, ‘safe use’ is a relative term in the time domain and any adverse effect may crop up even after several decades of use.

Carbon nanomaterials have emerged as alternate biocompatible materials. The fundamental queries regarding the properties of these carbon nanomaterials lie in their safe in vivo applications.^9,10 However, to waive off the toxicity menace of these nano-materials, the demonstration of the interaction of carbon nano-materials with a bio-system for an extended period of time is required. Humans explore newer rituals and in these processes have gained enough observation. In search of such a ritual we focused on the food roasted on an open fire. Barbecue remains synonymous to roasted food since the inception of fire in the Mesolithic age where roasting on an open fire was invariably associated with some charring on the uneven surface of meat (Fig. 1). Therefore, our investigations were focussed on procedures such as the barbeque. In addition, we extended our search for nano carbon materials in plant charcoal present in ‘Colic Calm’ branded gripe water (NDC 13992-001-01), USA, which is generally used for stomach ailments in infants.¹¹

Fig. 1 The charred parts (yellow circles) on the surface of roasted chicken.

Roasted meat is one of the preferred delicacies with a smoky aroma. Here, we show that the charred parts of the roasted meat contain graphene in the form of graphene oxide along with nano carbon particles. Interestingly, elemental analyses of such carbogenic materials show the presence of an appreciable quantity of nitrogen. Under mild treatment with dilute mineral acid the graphene oxide aggregates undergo layer separation yielding heteroatom doped graphene oxide (GO) sheets. The presence of graphene oxide in the charred parts of animal proteins suggest that no special conditions are required for its synthesis as considered earlier.¹² Earlier the formation of only nano carbon particles but no graphene have been shown during the pyrolysis of vegetable materials.^13,14 We specifically explored the content of plant charcoal that is added in the medically approved ‘Colic Calm’ branded baby gripe water. Such charcoal is used to achieve relief from gas, indigestion and abdominal distension with cramping pains in infants.¹¹ To our surprise we could identify the presence of graphene in Colic Clam.

2. Methods

A Charred part experiment

In a typical experiment, freshly prepared dressed chicken procured from the local market was used directly for roasting on an open fire without the application of external oil, butter or spices to simulate the mesolithic period of cooking. After roasting, the charred parts were mechanically separated out from the surface of the roasted chicken, crushed in a mortar–pestle and sieved to separate the carbon powder from any adhered cooked meat. The separated charred part was divided into two parts.

Experiment 1. One part of the charred material was chewed in the mouth for several minutes to allow salivary enzymes to work and then the mass was transferred into a beaker. The slurry mass was washed with distilled water twice and finally 20 mL HCl (pH = 2) was added into it to simulate stomach pH. The mixture was stirred at 50 rpm speed for 3 h followed by sonication for 15 min and finally the dispersed material was allowed to settle. The liquid containing the dispersed carbon was filtered using a cellulose acetate membrane filter of 0.1 μm pore-size and the carbon mass was washed with distilled water to collect it as digested charred carbon and subjected to characterization.

Experiment 2. The charred powder was repeatedly washed by a Soxhlet extractor with petroleum ether followed by acetone to remove any soluble organic matter formed during the roasting of the meat. The washed charred carbon was dried in air and labelled as ‘raw charred carbon’. The raw charred carbon was treated in HNO₃ : H₂O (1 : 1) for 3 h at room temperature and the excess acid was removed by the evaporation of the mixture using a boiling water bath.¹⁵ The obtained yellow-brown mass was dried in air and termed as ‘treated charred carbon’.

B Baby's gripe water

10 mL of branded Colic Calm baby's gripe water was removed after shaking the bottle well. The content was centrifuged and the residue was washed repeatedly three times with distilled water. The washed charcoal was dried in air as ‘raw charcoal’. For acid treatment of the raw charcoal a similar procedure was followed as described above, and the obtained mass was termed as ‘treated charcoal’.

3. Characterization

The field emission scanning electron microscopic (FESEM) study was carried out using a SURA 40VP field emission scanning electron microscope (Carl Zeiss NTS GmbH, Oberkochen, Germany) in high vacuum mode operated at 10 kV. Transmission electron microscopy (TEM) analysis was carried out using a EI Technai 20 U twin transmission electron microscope operated at 200 kV using a 400 mesh size carbon coated copper grid to deposit sample procured from Electron Microscopy Sciences, Hatfield, PA. For FESEM and TEM images the samples were dispersed in isopropanol. Powder X-ray diffraction (PXRD) was recorded on a Bruker AXS diffractometer with Cu-Kα (λ = 1.54 Å) radiation using PANalytical X'Pert HighScore Plus software. Raman spectra were recorded on a Raman spectrometer, WITEC MODEL, with 514.5 nm laser excitation. Elemental analyses for carbon, hydrogen and nitrogen were performed using a Perkin-Elmer 2400 micro-analyzer.

4. Results

The charred carbon was chewed in the mouth to allow the interaction with the enzymes present in saliva. The chewed carbon instead of swallowing was taken out and treated with dilute HCl pH (∼2) to mimic digestion in the stomach.¹⁶

1 Chewed charred part

Salivary glands in the mouth secrete an array of enzymes and substances mainly lingual lipase, amylase, mucin and lysozyme to pre-treat the food to facilitate digestion.¹⁶ The charred carbonaceous part from meat was chewed to allow its interaction with the saliva, and the saliva treated carbon mass was subjected to acid hydrolysis, as shown schematically in Fig. 2.

Fig. 2 Representation of the reaction to mimic the effect of acid on the charred parts of meat in the stomach.

The FESEM of the washed residue of the chewed part termed as ‘digested charred carbon’ after acid hydrolysis is shown in Fig. 3a. It confirms the presence of a sheet type structure of carbon along with nano particles. Fig. 3b shows the high resolution image of the white dotted area in Fig. 3a. The TEM image (Fig. 3c) corroborates the presence of thin sheet type structures similar to the FESEM image. The black dots present in the TEM image are due to the presence of carbon nano particles. The Raman spectrum of the digested chewed part shows the appearance of D- and G- bands at 1357 and 1570 cm⁻¹ due to the presence of sp³ and sp² hybridized carbon, respectively (Fig. 3d).

Fig. 3 Acid digested chewed charred carbon (a) FESEM image. (b) High resolution FESEM image of the area marked in (a). (c) TEM image and (d) Raman spectrum.

The characterization of saliva treated charred parts after acid hydrolysis led to sheet type structures with sp² and sp³ carbon atoms in the network, which were well separated on acidic treatment.

2 Treated charred part

The FESEM image (Fig. 4a) of the raw charred carbon shows the presence of multi-layered graphene sheets under turbostratic state along with carbon nanoparticles. Elemental analyses (Table 1) of the raw charred carbon show the presence of N, around 9.53%, suggesting that these carbons are doped with a high percentage of nitrogen atoms. The corresponding TEM image (Fig. 4d) confirms the presence of the multi-layered sheet type structure. Fig. 4b and c are the FESEM images of the treated charred carbon showing the presence of semi-transparent to transparent graphene oxide (GO) sheets of large area along with carbon nanoparticles. The GO sheets shown in Fig. 4c are transparent and visible only due to its appearance in a crumple form. The transparent GO sheets appear to create a blanket cover over the carbon nano particles (Fig. 4c). The TEM image of the treated charred carbon shown in Fig. 4e confirms the presence of transparent GO sheets along with carbon nanoparticles (dense part below the triangular area). The lightest part in the triangular area of Fig. 4e shows the presence of a single layer of GO sheet. The SADP of the treated charred carbon (Fig. 4f) is characteristic of a carbon structure with hexagonal symmetry. The inset of Fig. 4f shows the diffracted intensity of bright spots in SADP along the line. The SADP with a brighter diffraction intensity of the spots in the inner circle is characteristic of the single layered graphene (Fig. 4f and inset).¹⁷ Therefore, the FESEM, TEM and SADP of the treated charred carbon support the separation of the layers of turbostratic graphene sheets simply by treatment with dilute nitric or hydrochloric acid. The presence of heteroatoms in the turbostratic state readily separates the layers under dilute acid treatment.

Fig. 4 FESEM and TEM images of: (a and d) raw charred carbon; (b, c and e) treated charred carbon. (f) SADP of (e). Inset of (f) is the diffracted intensity taken along the line.

Table 1 Analysis of charred parts of meat and plant charcoal

	Elemental analysis of different samples
	%C	%N	%H	%O^a
a Calculated by difference.
Raw charred part	55.84	9.44	4.16	30.56
Treated charred part	48.94	9.53	4.31	37.22
Raw charcoal	89.44	1.02	7.59	1.95
Treated charcoal	83.36	1.92	8.03	6.69

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In the PXRD of raw charred carbon (Fig. 5a), the peaks at 2θ = 21.3, 28.44, 29.43 and 31.72° correspond to the d-spacing = 4.17, 3.13, 3.02 and 2.82 Å, respectively. The d-spacing of 4.17 Å is indicative of a lower degree of crystallization¹⁸ and the higher interplanar distance related to the stacking of disordered graphene sheets.¹⁸ In the PXRD of treated charred carbon (Fig. 5b), the peaks at 2θ = 14.38, 20.80, 26.67, 29.50 and 30.28° correspond to the d-spacing of 6.17, 4.26, 3.34, 3.02 and 2.95 Å, respectively. Here, the new peaks at 2θ of 14.38 and 14.92° were observed on high resolution of the red circled area, as shown in the inset of Fig. 5b, and correspond to the d-spacing of 6.17 and 5.93 Å, respectively. The higher d-spacing (6.17, 5.93 and 4.26) confirms the separation of layers as independent sheets in the treated charred carbon supporting the FESEM and TEM studies, as shown in Fig. 4. The peaks at 14.38 and 14.92° are at different positions than the GO only peak position. The presence of heteroatoms in the sheets with a change in chemical structure might be responsible for such shifting in the peak positions.¹⁹

Fig. 5 PXRD of (a) raw and (b) treated charred carbon (the inset is the zoomed image of the red circled area). Raman spectrum of (c) raw and (d) treated charred carbon.

In the Raman study of the raw charred carbon (Fig. 5c), the characteristic D, G and 2D bands are positioned at 1345, 1586 and 2695 cm⁻¹, respectively. For the treated charred carbon (Fig. 5d), the D, G and 2D bands are at 1367, 1590 and 2730 cm⁻¹ respectively, which are shifted toward the higher wave number when compared with those of the raw charred carbon. The 2D band in the treated charred carbon is unexpectedly broad with the maxima at 2730 cm⁻¹. This broadening is because of the fluorescent nature of the treated charred carbon due to the presence of fluorescent GO and carbon nano particles (not shown).

3 Charcoal from baby's gripe water

Colic Calm branded baby's gripe water contains black suspended plant charcoal (Fig. 6a). Fig. 6b is the FESEM image of the raw charcoal, in which the sheet type structure marked with a white dotted line and nearby ribbon type structures were due to disordered (turbostratic) graphene oxide. The arrow shows an indentation present on the brass stub used for the study, where a part of the graphene oxide sheet was deposited. The TEM image of raw charcoal (Fig. 6d) confirms the presence of a graphene oxide sheet. The disordered layers of graphene oxide present in raw charcoal upon dilute acid treatment undergo separation to yield crumbled GO sheets, as shown in Fig. 6c. The TEM image of treated charcoal (Fig. 6e) confirms the presence of highly transparent GO sheets. The SADP (Fig. 6f) of Fig. 6e displays the typical six-fold symmetry of graphene oxide. Twin spots in this SADP suggest an orientation mismatch at the folded site in Fig. 6e. The higher intensity of the inner circle spots in the SADP supports the presence of a single layer of GO.¹⁷ Along with a graphene structure, amorphous carbon and polyhedral shaped carbon structures are also present in the raw and treated carbon, respectively (Fig. S1 and S2†). The elemental analyses of raw and treated charcoal (Table 1) from gripe water confirms the presence of nitrogen in trace amounts, suggesting that turbostratic graphene from plant sources contains less nitrogen as it is mainly based on cellulose material in comparison with the turbostratic graphene from meat, which is enriched in proteins.

Fig. 6 (a) Gripe water. FESEM and TEM image of: (b and d) raw charcoal, and (c and e) treated charred carbon, respectively. (f) SADP of (e).

Fig. 7a is the PXRD of raw charcoal (from gripe water) having peaks at 2θ of 8.61, 16.50 and 22.65°, which corresponds to the d-spacing of 10.20, 5.37, and 3.92 Å, respectively. In the PXRD of treated charcoal, the peaks shifted towards the lower angle at 2θ of 8.55, 15.92 and 22.16° corresponding to the d-spacing of 10.33, 5.55 and 4.00 Å, respectively (Fig. 7b). The shifting of the PXRD peaks suggests an increase in the d-spacing and the presence of loosely bound layers in the raw charcoal, which upon acid treatment undergo separation, as shown in Fig. 6(c and e). The much higher inter-layer spacing in the raw plant charcoal suggests the presence of spaced graphene sheets in the plant charcoal, as suggested earlier.^20–25

Fig. 7 The PXRD of (a) raw and (b) treated charcoal. Raman spectrum of (c) raw and (d) treated charcoal.

In the Raman study of the raw charcoal (Fig. 7c), the characteristic D and G bands were positioned at 1335 and 1587 cm⁻¹, respectively, which shift to a higher wave number and are positioned at 1347 and 1598 cm⁻¹ for treated charcoal (Fig. 7d). Such shifts are similar to that observed previously for charred carbon. The 2D band is similar, as reported earlier.^26,27

5. Discussion

Meat contains mainly proteins, which upon heating undergo Maillard caramelization and polymerization reactions to produce heteroatom containing cyclic compounds.^28–34 Under charring conditions, the five membered hetero rings may rearrange to six membered rings³⁵ followed by condensation to generate graphene units doped with heteroatoms at the edges and basal planes for stability.^35–37 In addition, peripheral oxo groups are introduced at the edges of the graphene layer during carbonization in air.²⁰ The heteroatoms at the edges or in the basal planes mismatch the standard interlayer spacing in graphite to yield the turbostratic form.²⁰ The elemental analyses of the charred materials of meat and plant charcoal are tabulated in Table 1. The presence of a significant amount of N supports the presence of nitrogen doped graphene oxide sheets especially from charred meat. However, note that the materials subjected to elemental analysis does contain carbon nano particles in part and therefore the absolute value of nitrogen analysis should be taken with caution and the possibility for the existence of the nitrogen doped carbon nano particles may not be ruled out.

The disordered state of graphene in plant charcoal could be related to the formation of turbostratic graphene similar to the case of natural fire in forests.^21–25 The interplanar distance of graphite is not achieved because of the formation of heteroatom functionalities, which prevents the close packing of sheets.²⁰ The formation of a graphitic structure requires a temperature of around 3500 °C,²⁰ which is significantly higher than the temperature used in roasting the meat and also in the synthesis of plant charcoal. The interaction of chewed parts with dilute HCl is not ideally akin to the natural process in digestion but provides a close chemistry regarding the fate of the charred parts in the human stomach. The FESEM, TEM of the chewed charred part after standard cleaning (Fig. 3a–c) confirms the presence of a layer of graphene sheets doped with heteroatoms, as confirmed by the elemental analyses (Table 1). The graphene sheets present in loose bundles are formed due to heteroatom incorporation, which are separated out readily to single layer GO sheets upon treatment with dilute HNO₃ or HCl acid, as shown in Fig. 4 and 6(c and e). The intensity of the bright spots in SADP (Fig. 4 and 6(f)) confirms the presence of a single layer of GO sheet.¹⁷ Such single layer sheet separation of hetero GO sheets on treatment with dilute acid is noteworthy because the synthesis of GO from graphite requires laborious and harsh acidic conditions.^19,38–41

6. Conclusion

The practice of barbecuing food can be traced back to the discovery of fire. This study revealed the existence of heteroatoms doped graphene oxide with carbon nano particles in food prepared by barbecuing. Surprisingly, graphene derivatives and carbon nano particles are also present in plant charcoal used in brand medicine approved for infants to cure stomach ailments. The evolution of humans since the Mesolithic age parallel to the discovery of fire has continued without showing any ill effect caused by the intake of such an inadvertent contaminant in roasted food. If mutation occurred under the stress of such food intake in human, our evolution has acquired full immunity against the use of such nano carbon materials.

Acknowledgements

M.S. acknowledges the University Grant Commission, India for Dr D. S. Kothari Post Doctoral Fellowship. S.S. acknowledges Cromoz Inc., USA for financial support for the study and a Ramanna Fellowship from DST, New Delhi to sustain research. Both the authors thank Master Sagneic Biswas of Boston, USA for loaning Colic Calm gripe water used in this study.

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Footnote

† Electronic supplementary information (ESI) available: S1, S2 SEM image of raw and treated charcoal. See DOI: 10.1039/c4ra04022h

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