Biomineralisation by earthworms: an investigation into the stability and distribution of amorphous calcium carbonate

Background Many biominerals form from amorphous calcium carbonate (ACC), but this phase is highly unstable when synthesised in its pure form inorganically. Several species of earthworm secrete calcium carbonate granules which contain highly stable ACC. We analysed the milky fluid from which granules form and solid granules for amino acid (by liquid chromatography) and functional group (by Fourier transform infrared (FTIR) spectroscopy) compositions. Granule elemental composition was determined using inductively coupled plasma-optical emission spectroscopy (ICP-OES) and electron microprobe analysis (EMPA). Mass of ACC present in solid granules was quantified using FTIR and compared to granule elemental and amino acid compositions. Bulk analysis of granules was of powdered bulk material. Spatially resolved analysis was of thin sections of granules using synchrotron-based μ-FTIR and EMPA electron microprobe analysis. Results The milky fluid from which granules form is amino acid-rich (≤ 136 ± 3 nmol mg−1 (n = 3; ± std dev) per individual amino acid); the CaCO3 phase present is ACC. Even four years after production, granules contain ACC. No correlation exists between mass of ACC present and granule elemental composition. Granule amino acid concentrations correlate well with ACC content (r ≥ 0.7, p ≤ 0.05) consistent with a role for amino acids (or the proteins they make up) in ACC stabilisation. Intra-granule variation in ACC (RSD = 16%) and amino acid concentration (RSD = 22–35%) was high for granules produced by the same earthworm. Maps of ACC distribution produced using synchrotron-based μ-FTIR mapping of granule thin sections and the relative intensity of the ν2: ν4 peak ratio, cluster analysis and component regression using ACC and calcite standards showed similar spatial distributions of likely ACC-rich and calcite-rich areas. We could not identify organic peaks in the μ-FTIR spectra and thus could not determine whether ACC-rich domains also had relatively high amino acid concentrations. No correlation exists between ACC distribution and elemental concentrations determined by EMPA. Conclusions ACC present in earthworm CaCO3 granules is highly stable. Our results suggest a role for amino acids (or proteins) in this stability. We see no evidence for stabilisation of ACC by incorporation of inorganic components.

Effect of high pressure homogenisation and heat treatment on physical properties and stability of almond and hazelnut milks

The effect of high pressure homogenisation (HPH) and heat treatments on physicochemical properties and physical stability of almond and hazelnut milks was studied. Vegetable milks were obtained and homogenised by applying 62, 103 and 172 MPa (MF1, MF2 and MF3, respectively). Untreated and MF3 samples were also submitted to two different heat treatments (85 °C/30 min (LH) or 121 °C/15 min (HH)). Physical and structural properties of the products were greatly affected by heat treatments and HPH. In almond milk, homogenised samples showed a significant reduction in particle size, which turned from bimodal and polydisperse to monodisperse distributions. Particle surface charge, clarity and Whiteness Index were increased and physical stability of samples was improved, without affecting either viscosity or protein stability. Hazelnut beverages showed similar trends, but HPH notably increased their viscosity while change their rheological behaviour, which suggested changes in protein conformation. HH treatments caused an increment of particle size due to the formation oil droplet-protein body clusters, associated with protein denaturation. Samples submitted to the combined treatment MF3 and LH showed the greatest stability.