Wheat bran extracts as biomineralization scaffolds: An exploratory study leading to aqueous solution synthesis of spheroidal brushite particles

Journal Pre-proof Wheat bran extracts as biomineralization scaffolds: An exploratory study leading to aqueous solution synthesis of spheroidal brushite particles ´ Jorge Luis Zavala-Corrales Rene´ Renato Balandran-Quintana Jose´ Antonio Azamar-Barrios Ana Mar´ıa Mendoza-Wilson Paola ´ ´ Sergio Pompa-Redondo Guadalupe Hurtado-Solorzano Jesus

PII:

S0960-3085(19)31023-5

DOI:

https://doi.org/doi:10.1016/j.fbp.2020.03.003

Reference:

FBP 1232

To appear in:

Food and Bioproducts Processing

Received Date:

18 October 2019

Revised Date:

8 February 2020

Accepted Date:

11 March 2020

´ Please cite this article as: Zavala-Corrales, J.L., Balandran-Quintana, R.R., Azamar-Barrios, ´ J.A., Mendoza-Wilson, A.M., Hurtado-Solorzano, P.G., Pompa-Redondo, J.S.,Wheat bran extracts as biomineralization scaffolds: An exploratory study leading to aqueous solution synthesis of spheroidal brushite particles, Food and Bioproducts Processing (2020), doi: https://doi.org/10.1016/j.fbp.2020.03.003

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Wheat bran extracts as biomineralization scaffolds: An exploratory study leading to aqueous solution synthesis of spheroidal brushite particles

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Jorge Luis Zavala-Corralesa,b

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René Renato Balandrán-Quintanaa*

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José Antonio Azamar-Barriosc

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Ana María Mendoza-Wilsona

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Paola Guadalupe Hurtado-Solórzanoa

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Jesús Sergio Pompa-Redondoa

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a

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de Alimentos de Origen Vegetal. Carretera Gustavo Enrique Astiazarán Rosas, No. 46.

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83304. Hermosillo, Sonora, México.

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Formerly MSc Student at Centro de Investigación en Alimentación y Desarrollo, A.C.

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Centro de Investigación en Alimentación y Desarrollo, A.C. Coordinación de Tecnología

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Unidad Mérida. Departamento de Física Aplicada. Carretera antigua a Progreso Km. 6.

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97310. Mérida, Yucatán, México.

CINVESTAV-IPN-Mérida. Centro de Investigación y de Estudios Avanzados del IPN,

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*Corresponding autor: Tel. +52 662 2892400x431. E-mail: [email protected]

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Abstract

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Biomineralization in solution was investigated with aqueous extracts of wheat bran, pH 5-

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8, added with CaCl2 and incubated 5, 10, or 15 d at 6±2 °C. Solution pH and weight of

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precipitates were monitored. Particles were isolated and characterized by stereoscopic

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microscopy, scanning electron microscopy (SEM), X-ray energy dispersive spectroscopy

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(EDS), Fourier transform infrared spectroscopy (FTIR), and X-ray powder diffraction

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analysis (XRD). Stereoscopical images showed polydisperse particles of undefined shape at

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5 d in the whole range of pH and CaCl2 concentrations. Starting from 10 d, the effects of

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pH and CaCl2 concentration were evident on precipitate weight, particle size, and

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morphology, as smooth spherical particles were seen. Best conditions for production of

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particles were incubating 10 d, initial pH 6.27, 0.75 M CaCl2. SEM images revealed

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spheroidal particles, 110-200 m diameter, with internal microstructure consisting of

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elongated sheets, 200 nm width, 20 nm thick, radially aligned. The P/Ca rates were around

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1.0, whereas FTIR and XRD analyses evidenced a single phase of calcium phosphate

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dihydrate (Ca2HPO4∙2H2O), or brushite, in a range of final pH 5.3-6.3. Results confirmed

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that wheat bran extracts act as scaffolds for biomineralization in aqueous solution.

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Keywords: in vitro biomineralization; sustainability; cereal by-products; calcium phosphates; spherical brushite

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Highlights:

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 Lyophilized wheat bran extracts were reconstituted in water

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 Extracts were tested as scaffolds for biomineralization

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 At final pH 5.3 - 6.3, spheroidal brushite particles were synthesized

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 Phosphate precursor ions come from the wheat bran extracts

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1. Introduction

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Brushite or calcium hydrogen phosphate dihydrate (CaHPO4∙2H2O) is a biomineral formed

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in the guano of bat caves, and found in urinary stones (Bhojani, Jethva, Joshi, 2019; Frost

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and Palmer, 2011; Zhang, Wang, Putnis, 2019). Brushite prepared in vitro has value as soil

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fertilizer, feed additive, abrasive in toothpastes, cement for bone graft and dental implants,

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drug delivery, cancer therapy, and development of biosensors (Tamimi, Sheikh, Barralet,

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2012; Toshima, Hamai, Tafu, Takemura, Fujita, Chohji et al., 2014). Due to its importance,

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elucidating formation mechanisms and controlling morphology of brushite are topical

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issues (Ferreira, Oliveira, Rocha, 2003; Rubini, Boanini, Bigi, 2019). Among calcium

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phosphates (CaP) brushite is the most stable phase at pH <6.5 so can be easily obtained by

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aqueous solution synthesis (Boistelle and Lopez-Valero, 1990). Polymer-based scaffolds

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are also used to precipitate brushite (Gashti, Bourquin, Stir, Hulliger, 2013; Suryawanshi

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and Chaudhari, 2014).

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Morphology of brushite is diverse. Depending on pH, precursor concentration, and

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preparation method, water lily-, flat-plate-, petal-like-, or flower-like-shaped crystals are 3 Page 3 of 36

obtained (Toshima et al., 2014; Brundavanam, Poinern, Fawcett, 2014; Dabiri, Lagazzo,

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Barberis, Farokhi, Finochio, Pastorino, 2016; Lim, Kassim, Huang, Khiewc, Chiu, 2009;

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Mandel and Tas, 2010). Growth of CaP crystals, including brushite, often results in

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particles with sizes within the nanometer to millimeter scale and varied shapes. Spherical

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shape is of major interest because of several advantages regarding dental and orthopedic

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applications, but is the most difficult to produce as well (Bohner, Tadier, van Garderen, de

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Gasparo, Dobelin, Baroud, 2013).

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Spherical particles of brushite can be obtained by template methods such as reverse

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microemulsion (diameter 23-87 nm) (Singh, Singh, Aggarwal, Mandal, 2010), and cement

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past emulsion (diameter 200-1,000 m) (Moseke, Bayer, Vorndran, Barralet, Groll,

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Gbureck, 2012). Spheroidal brushite granules (diameter 500-600 m) have also formed in

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recovering P from industrial effluents (Caddarao, Garcia-Segura, Ballesteros, Huang, Lu,

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2018). These methods are some bit laborious and crystal arrangement inside the particles is

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irregular.

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At the best of our knowledge, there are no studies about biomineralization in which

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agroindustrial by-products are used as scaffolds. The recovery of CaP from dairy co-

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products has been studied because of the fouling of membranes and heat exchangers caused

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by precipitation of these salts (Mekmene, Quillard, Rouillon, Bouler, Piot, Gaucheron,

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2009). In other work, the formation of some CaP phases during cold gelation-desolvation of

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proteins present in aqueous wheat bran extracts was informed (Luna-Valdez, Balandrán-

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Quintana, Azamar-Barrios, Ramos Clamont-Montfort, Mendoza-Wilson, Mercado-Ruiz et

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al., 2017), and this led us to hypothesize that such extracts could serve as scaffolds for

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biomineralization in aqueous solution under controlled conditions. The objective of the

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present work was to verify this hypothesis. Results indicate that biomineralization of wheat

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bran extracts could be an alternative to the synthesis of spheroidal brushite particles. As

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wheat bran is majorly intended to animal feed, P contained in it often goes to water sources,

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contributing to eutrophication. Thus, a sustainable process is anticipated since P from wheat

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bran is recovered and spheroidal brushite forms at the same time. Extracted solids from

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wheat bran could be then intended to animal feed.

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2. Materials and Methods

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Wheat bran (Triticum aestivum L.) was obtained from a commercial mill. Chemicals were

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from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise indicated. Milli-Q water (18

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MΩ) was used to prepare solutions, as well as to wash and extract wheat bran. General

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experimental strategy is depicted in Scheme 1 and explained below.

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2.1. Obtaining Aqueous Extracts of Wheat Bran (AEWB)

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Lyophilized AEWB was obtained according to Campas-Ríos, Mercado-Ruiz, Valdéz-

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Covarrubias, Islas-Rubio, Mendoza-Wilson, Balandrán-Quintana (2012) and Luna-Valdez

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et al. (2017). Briefly, wheat bran was subjected to washing, drying, and aqueous extraction

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(3 h, 1:10 w/v ratio), after which insoluble solids were removed by filtration and pH

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adjusted to 8.0 with NaOH. Filtrate was identified as non-lyophilized AEWB (NL-AEWB)

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and its soluble protein content was estimated by Bradford method (Bradford, 1976). NL-

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AEWB was left inside refrigerator until collecting extracts from several batches, which

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were frozen and lyophilized to recover a powder, identified as lyophilized AEWB (L-

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AEWB). A straight process to obtain L-AEWB was also performed, consisting of adjusting

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pH of NL-AEWB to 8.0 after extracting each batch, and freezing immediately; then several

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batches were lyophilized. Total protein content of L-AEWB was determined by

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microkjeldahl method (AOAC, 1990).

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2.2. Biomineralization in Aqueous Solution

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2.2.1. Preliminar Trials

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Water dispersibility of L-AEWB and weight of humid precipitates (g/100 g of L-AEWB)

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after 15 d incubation at 6±2 °C were criteria to determine experimental concentrations of L-

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AEWB and biomineralization times. Another independent variable was a heat treatment in

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water bath (68.5 °C, 3 h), given to water-reconstituted L-AEWB, followed by cooling to 25

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°C before biomineralization. This heat treatment was applied because it constituted a step

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in the previous work of Luna-Valdez et al. (2017) in which mineral phases were found

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during cold gelation-desolvation of AEWB proteins, so the question arose about if heat

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treatment had to do with the biomineralization process. For preliminary trials, pH of

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reconstituted L-AEWB was 6.27, i.e., that resulting after aqueous dispersion. Trials were

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performed according the procedure outlined in section 2.2.2.

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2.2.2. Biomineralization

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The effects of concentrations of both L-AEWB and CaCl2, incubation time, initial pH, and

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a heat pre-treatment given to L-AEWB, on characteristics of mineral phases, were

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recorded. Levels of some variables were determined as explained in section 2.2.1.

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Concentration range of CaCl2 was 0.25-1.5 M because this was the one in which mineral

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phases were found in the work of Luna-Valdez et al. (2017). The only source of P in this

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study was the AEWB. Biomineralization was started by pouring into test tubes 2 mL of

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reconstituted L-AEWB, subjected or not to heat pre-treatment in water bath (68.5 °C, 3 h),

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plus 0.2 mL of 0.25, 0.75, or 1.5 M CaCl2. The mix was immediately vortexed. All tubes

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were incubated during 5, 10, or 15 d at 6±2 °C. In order to both retain all precipitate and

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obtain a clear particle-free supernatant, samples were centrifuged (5,000xg, 5 min) after

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each period. After completely pouring the supernatants, weight of humid precipitates was

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recorded and expresed as g/100 g of L-AEWB to have a gross estimation of process yield.

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Then, precipitates were thoroughly washed with Milli-Q water to get rid soluble material.

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Insoluble particles were recovered by decantation, dried at 40 °C overnight, and

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characterized. On the basis of work from Luna-Valdez et al. (2017), it was assumed that

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particles consisted of one or more mineral phases, so that from now on the term ‘mineral

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phase’ will be used when referring to particles. After characterization, a scaled experiment

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was conducted by triplicate (adding 10 mL of CaCl2 into 100 mL of L-AEWB 10% w/v),

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under experimental conditions which were considered to be the best, in order to calculate

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the process yield in terms of mineral phase.

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2.2.3. Verification of pH throughout Biomineralization

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The pH to which biomineralization took place was determined by conducting an

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experiment at 3 different initial pH (i.e., pH prior adding CaCl2). The resulting pH when

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dissolving the L-AWEB was 6.27, and this was adjusted to 5 or 8 with either HCl or

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NaOH, so that the initial pH range under study was 5, 6.27, and 8. Biomineralization was

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performed with 10% w/v L-AEWB obtained by the straight process (this process of

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obtaining L-AEWB is outlined in Scheme 1), by addition of CaCl2 0.75 M (1:10 v/v) and

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incubating 10 d at 6±2 °C. pH was recorded daily with a pH-meter, with no stirring. In

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order to have a negative control (basic pH), an additional run was performed adjusting

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initial pH to 11 by adding NaOH, with no daily record of pH. Precipitated mineral phases

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were recovered as explained in section 2.2.2, and characterized as follows.

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2.3. Characterization of Mineral Phases

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Diameters of fifty individual particles, observed under a stereomicroscope (Leica

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Microsystems, Wetzlar, DEU) were analyzed in the OriginPro 8.6.0 software (OriginLab

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Corp., MA, USA) to determine particle size distribution and mean diameters. Detailed

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morphology and internal microstructure of samples coated with a gold-palladium layer into

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a Q150R ES bombardment coater (Quorum Technologies Ltd., USA) were observed by

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SEM in a JEOL JSM-7600F microscope (JEOL Ltd., Tokyo, JPN), at magnifications of

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700 ̶ 10,000x, 2.0 kV. Elemental composition was analyzed by EDS in the electron

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microscope, by scanning variable areas (30,000, 853, or 15 μm2) of samples coated with a

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gold-palladium layer. CaCO3, SiO2, GaP, Wollastonite, MgO, KCl and MAD-10 Feldspar

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were used as reference materials for detector calibration. Data of elemental composition

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were reported as both weight % and atomic %.

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Functional groups were analyzed by FTIR in a Nicolet Nexus 670-FTIR equipment

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(Nicolet Instrument Corp., Madison, WI, USA). Samples were pulverized and

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homogenized with KBr. Sixty four scans were performed per sample, resolution 4 cm-1,

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speed 0.6329 cm/s, wavenumber range 400-4000 cm-1. Identity of mineral phases was

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verified by XRD in a Bruker D-8 Advance diffractometer, wavelength 1.54 Å, 2 from 10

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to 70 °, 0.5 s per step, step size 0.02°, 40 kV, 30 mA.

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It was assumed that protein could be present in mineral phases, so a representative

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sample (150 mg) was demineralized by stirring 90 min in 0.5 N HCl, followed by dialysis

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in centrifuge microcones (Sigma). SDS-PAGE was performed in a Mini Protean Tetra Cell

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(Bio-Rad Laboratories Inc., Hercules, CA), 12% gels, 14 mA, 2.5 h. Chemicals for SDS-

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PAGE were from Bio-Rad. Gels were documented in a Gel DocTM XR + System (Bio-

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Rad).

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2.4. Experimental Design and Statistical Analysis

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Experimental design was completely randomized. Effects of independent variables were

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analyzed through an ANOVA in the NCSS software (Hintze, 2007). Means analysis was

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made by the Tukey-Kramer test (p<0.05). Morphology and crystallinity were not evaluated

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statistically because being qualitative variables.

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3. Results and Discussion

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3.1. Mineral Phase Yields

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Concentrations higher than 10% w/v made L-AEWB difficult to disperse, so concentrations

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of 2.5, 5.0, and 10% w/v, with addition of 1.5 M CaCl2 were explored to observe effects of

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heat pre-treatment on precipitate weight after 15 d. When no heat treatment was applied to

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L-AEWB, the more concentrated the L-AEWB the higher the precipitate weight, whereas

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the heat treatment resulted in a lower weight regardless the L-AEWB concentration (Fig.

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S1).

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These results were the basis to perform further biomineralization assays with 2.5 ̶ 10%

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w/v L-AEWB, and incubating 5-15 d at 6±2 °C. Despite lower yields obtained with L-

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AEWB subjected to heat treatment, biomineralization experiments were also carried out

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under this condition to observe its effect on type and morphology of mineral phases.

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Fig. 1 shows the effects of CaCl2 concentration and time on precipitate yield, in terms of

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g of precipitate by 100 g of L-AEWB 10% w/v not subjected to heat pre-treatment, after

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biomineralization. In general, the higher the CaCl2 concentration the higher the precipitate

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yield. PO ̶ concentration is constant at each time in Fig. 1, since is the one naturally present

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in the L-AEWB solution. At the lowest concentration of Ca2+ (0.25 M), precipitate yield

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was unchanged in the course of 15 d. Here, without measuring, we could reasonably think

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that either Ca2+ or PO ̶ were the limiting ions and that one or the other could have been

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consumed since 5 d. At a higher concentration of Ca (0.75 M) yield is higher as well,

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indicating that more Ca2+ was necessary since PO

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differences in the course of 15 d are seen so is suspected that at 0.75 M, Ca2+ is exhausted

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as early as 5 d. At 1.5 M CaCl2, PO ̶ concentration is still constant, but yield was more than

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triplicated either at 5, 10 or 15 d. This indicates that PO was not the limiting ion or

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otherwise larger precipitates were no longer formed. Currently work is in progress to

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explore reaction kinetics in depth.

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remained constant; however, little

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As the objective of the present work was to explore L-AEWB as potential scaffold for

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biomineralization, and because precipitate yield was not so different between 10 and 15 d,

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in order to save time and optimize resources it was decided to run subsequent assays by

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adding 0.75 M CaCl2 to reaction media and incubating 10 d at 6±2 °C.

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3.2. Particle Morphology, as Affected by Heat pre-Treatment, Incubating Temperature, Adaptation to Extracting Method, and L-AEWB Concentration

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In general, stereoscopic observation of precipitates coming from biomineralization with

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preheated L-AEWB, showed spheroidal-shape particles (Fig. S2). In non pre-heated

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samples spheroidal particles were also observed, but in greater amounts (Fig. S2). Attempts

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to induce biomineralization at 25 °C were also made, but only particles with varied

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morphology, or agglomerated matter were observed (Fig. S2). The latter was due to 10 Page 10 of 36

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microbial growth, evidenced by off odors. No antimicrobial agents were used to preserve

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the extracts in order not to disturb their chemical composition. Nor did particles with a well-defined shape grow when, before freezing for

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lyophilization, pH of NL-AEWB was readjusted to 8.0. This pH re-adjustment was carried

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out because NL-AEWB was left inside the refrigerator for several days, until extracts from

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several batches were collected to lyophilize all them at the same time. However, chemical

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changes must have occurred in NL- AEWB during the cold period as the pH decreased to

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6.0; hence the decision to re-adjust it to 8.0. Such changes affected particle formation

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during biomineralization, even after pH re-adjustment or regardless if L-AEWB was

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subjected or not to heat treatment (Fig. S3).

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Because of the effects of cold period and/or pH re-adjustment on particle formation, an

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experiment was run with no adjustment of pH to 8.0, so NL-AEWB was lyophilized having

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pH 6.3, i.e., the natural pH after aqueous extraction of wheat bran. After biomineralization

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using this L-AEWB, with no heat treatment, a large number of particles with defined

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spheroid morphology, without lumps or agglomerations, was produced, as was seen in

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stereoscopical images (Fig. S4A).

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By the other hand, particles produced after biomineralization of L-AEWB obtained by

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the straight process had defined spherical morphology, without agglomerations, and larger

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than those formed when pH was not adjusted (Fig. S4B). In samples from L-AEWB not

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subjected to heat treatment, larger spheres were observed, compared to those obtained from

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L-AEWB with heat pre-treatment (Fig. S4B). Judging by the performance, size, and well-

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defined shape of particles, it was considered that the best L-AEWB with which to work in

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the future was that obtained by the straight process and not subjected to heat treatment.

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3.3. Effects of Initial pH and Time on both Appearance of L-AEWB Preps and pH after Adding CaCl2

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adding CaCl2, except in those with initial pH 5.0 (Fig. 3; Table 1). If, as expected, a

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calcium phosphate phase was forming, the pH decrease just after adding Ca2+ would

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indicate that H+ was released by dissociation of H2PO42- (Arifuzzaman and Rohani, 2004).

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In samples with initial pH 5, an average value of 4.9 was maintained during 10 d, and

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almost no particles were precipitated. At initial pH 6.27, pH dropped after adding CaCl2,

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and an average constant value of 5.33 was maintained over 10 d; here the highest amount of

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precipitate was recovered, with a yield of 63.9%. At initial pH 8, this value decreased after

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addition of CaCl2, but then was unchanged during 10 d, averaging 6.27 (Table 1); here, a

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little more precipitate was obtained compared to samples with initial pH 5.

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Spontaneous precipitation and significant decreases of pH occurred in all tubes after

Size and quantity of particles were affected by pH (Figure S5). The fewest amount of

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small particles were found at pH 5. The largest particles with well defined morphology

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were recovered at initial pH 6.27, while at pH 8 the particles were much larger than at pH 5

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but smaller than at pH 6.27. As expected, at pH 11 precipitate particles did not have any

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defined form. This results indicate that best particles in terms of morphology and size, were

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formed from L-AEWB obtained by the straight process and initial pH 6.27, but formed at

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pH 5.33 because of the pH drop after addition of CaCl2.

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3.4. FTIR Analysis

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Representative IR spectra of mineral phases obtained after biomineralization in solution

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with L-AEWB 10% w/v, are shown in Fig. 4A. Identical spectra can be seen either L-

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AEWB was subjected or not to heat pre-treatment. This indicates that, in response to heat 12 Page 12 of 36

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(68.5 °C), molecular structure of the scaffold that directs nucleation and growth, is not

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modified to such an extent that results in forming a different mineral phase. By comparison

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to IR spectra of calcium phosphates in literature, it was found that precipitated phase at

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initial pH 6.27-8.0 was calcium hydrogenphosphate dihydrate (CaHPO4∙2H2O) or brushite

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(Rubini et al., 2019). IR signals of brushite are well identified. Doublets at 3542-3480 cm-1 and 3278-3165

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cm-1 are attributed to asymmetric and symmetric stretching of water molecules which are

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loosely hydrogen-bonded, and to water molecules which experience a stronger hydrogen-

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bonding, respectively (Trpkovska, Šoptrajanov, Malkov, 1999). The strong band at 1649

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cm-1 corresponds to bending of water molecules. The remain zone of the spectrum belongs

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to vibrations of the HPO4 group. Band at 1209 cm-1 is assigned to the H-in-plane bending,

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and those at 1145, 1066, 986, and 874 cm-1 are due to the PO stretching. The medium band

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at 795 cm-1 accounts for H-out-of-plane bending, and those at 577 and 526 cm-1 are

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assigned to OPO bending. Bands at 2384 and 660 cm-1 do not represent fundamental

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vibrations of brushite but overtone and liberation, respectively (Singh et al., 2010).

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IR spectra also show that low quantities of protein could be present into the brushite

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structure. Shoulders at 1720 and 1576 cm-1, and the weak absorption band at 1450 cm-1

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could account for Amide I and Amide II groups, respectively, of proteins (Barth, 2007).

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Similar IR spectra of brushite were obtained when this was synthesized in presence of

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polyaspartic acid, assuming an interaction of the latter with the crystal (Rubini et al., 2019).

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3.5. XRD Analysis

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Signals in the X-ray diffractograms of Fig. 4B match that of CaHPO4∙2H20 (PDF 00-009-

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0077), confirming that pure brushite was precipitated at both final pH 5.3 and 6.3, i.e., the 13 Page 13 of 36

pH after 10 d (Table 1), which is very similar to the 5.13-5.96 range reported by Mekmene

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et al. (2009). The different intensities of peaks between samples coming from heat-treated

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or non-heat-treated L-AEWB, is probably due to different average longitude and aspect

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ratios of brushite crystals, as has been shown for other systems (Inoue and Hirasawa, 2013).

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Diffractograms in Fig. S6 indicate that both CaC2O4·2H2O (weddelite) and brushite were

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formed at initial pH 5, while no crystalline phases were found at pH 11.

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3.6. Morphology and Size of Particles

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Representative stereoscopical images of how particle formation was affected by CaCl2

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concentration, incubation time, and heat pre-treatment are shown in Fig. S7. Images were

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taken for the experiment with initial pH 6.27. In general, particles from precipitates isolated

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after different periods looked as spherical, with the lowest production at 5 d regardless the

320

concentration of CaCl2. At 0.25 M CaCl2, some bit irregular particles were seen at 10 d, but

321

their morphololgy changed at 15 d. At 0.75 M or higher, morphology was well defined and

322

surface looked like if smooth. According to particle size distribution (Fig. S8) size ranges

323

of 100-180 m and 100-200 m were found for particles coming from heat-treated or no-

324

heat-treated L-AEWB, respectively, the latter with a wider size distribution.

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The effects of both obtaining L-AEWB by the straight process and the heat pre-

326

treatment on particle morphology, are seen in SEM images of Fig. 5. Similar shapes are

327

obtained in the synthesis of water-lily-shaped (WL) crystals of brushite (using CaCO3 as

328

the Ca2+ source) in presence of small concentrations of Zn. At very low Zn concentrations,

329

individual plates are thickened, and a dumbbell-type structure is acquired. Increasing

330

concentrations of Zn result in denser dumbbells, which eventually turn into spherical

331

microgranules (Miller, Kendall, Jain, Larson, Madden, Tas, 2012). Presumably, Zn inhibits

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14 Page 14 of 36

the crystallization of brushite along its face (010), causing crystallization of aggregated

333

brushite crystals (Lundager Madsen, 2008), whereas spherical shape is acquired in response

334

to the gradual increase in surface free energy (Miller et al., 2012). Crude wheat bran has a

335

Zn content around 7.3 mg/100 g, frequently complexed to phytic acid (Brouns, Hemery,

336

Price, Mateo Anson, 2012). Although no Zn was detected in our system by EDS, the

337

minute concentrations of this ion reported as enough to change morphology from WL- to

338

dumbbell-shape, suggest the possibility that traces of Zn are in the brushite particles which

339

could be detected by atomic absorption spectroscopy. Nevertheless, plate thickness in our

340

particles is not so large as that reported by Miller et al. (2009), so probably a different

341

mechanism underlies sphere formation. Work is currently done to address this subject.

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SEM microphotographs of mineral phases from biomineralization of L-10% w/v

343

AEWB, at different initial pH are seen in Fig. 6. Particles formed at initial pH 5 had an

344

ovoid shape of 10x3.7x2.6 m in their long and two width dimensions, respectively. These

345

consisted of stacked plate-shaped crystals, with individual dimensions of 0.1 m thick, 0.2

346

m width; spherical particles of 10 m in diameter were distributed between the ovoids. As

347

discussed in section 3.5 (Fig. S6), a weddellite phase was detected by XRD at initial pH 5.

348

Weddellite is the calcium oxalate dihydrate (CaC2O4·2H2O), which by partial dehydration

349

converts to the monohydrate whewellite (CaC2O4·H2O). Although whewellite crystallizes

350

more

351

Thongboonkerd, 2017), its formation is inhibited by peptides and proteins, which are the

352

same factors that stabilize weddellite (Izatulina and Punin, 2012). Wheat bran has, in

353

average, 60.8 mg /100g (DM) of soluble oxalates, extracted with water at 80 °C

354

(Boontaganon, 2009). Some of these oxalates could have been be extracted at 5°C and be

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easily

than

weddellite

at

acid

pH

(Manissorn,

Fong-Ngern,

Peerapen,

15 Page 15 of 36

present in AEWB, favoring the formation of weddellite at pH 5 because of the presence of

356

proteins. At initial pH 6.27, spheroidal particles 70-90 m diameter are seen, which are

357

internally structured by radially aligned plates with individual dimensions 0.4-0.8 m width

358

and 0.08-0.2 m thick. This is a ledge structure reported for brushite under certain

359

conditions. Bilayers of brushite consist of calcium and phosphate sheets, separated by a

360

sheet of water (Flade, Lau, Mertig, Pompe, 2001). The ledge structure appears in presence

361

of osteocalcin, a protein that regulates hydroxyapatite formation (Flade et al., 2001), and is

362

also revealed after soaking of K- or Na-doped brushite crystals, in simulated body fluid

363

(SBF) (Tas and Bhaduri, 2004). Presence of osteocalcin, K or Na, inhibit brushite growing

364

and favors nucleation of carbonated apatite. In the present work, components yet unknown

365

of AEWB could be responsible for both the dumbbell- and ledge-like structures. At initial

366

pH 8, particles look as spheres, 95 m diameter, also consisting of radially aligned plate

367

shaped crystals; between some crystals something like a glue as well as scattered

368

amorphous matter, are observed. By the other hand, at initial pH 11 only agglomerated

369

material was precipitated (Fig. 6).

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Growth of brushite crystals is a surface reaction controlled process (Marshall and

371

Nancollas, 1969). That produced in vitro has diverse morphologies, depending on pH,

372

precursor concentration, and preparation method. If prepared by aqueous solution synthesis,

373

water lily-shaped crystals of brushite are obtained by mixing NH4H2PO4 and CaCO3, final

374

pH 5.9 (Mandel et al., 2010), whereas flat-plate-shaped crystals are prepared from a buffer

375

solution (KH2PO4+Na2HPO4) pH 7.5, added with CaCl2·2H2O, final pH 5.3 (Mandel et al.,

376

2010). Brushite can also be transformed from petal-like (nested structures at the center of

377

particles) into plate-like (parallelogram shape, stacked in multiple layers, 1-2 m thick) by

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increasing initial pH and/or HPO42− and Ca2+ concentration at room temperature (Toshima

379

et al., 2014). Flower-like brushite crystals can be grown by immersion of Mg sustrates in an

380

electrolyte consisting of Ca(NO3)2 and KH2PO4, pH 4.0, with 500-700 m in size after 180

381

min immersion, consisting of multi stacked plates (Brundavanam et al., 2014). Also,

382

brushite plate-like particles 50 μm length, 20 μm width, and 100-200 nm thickness, are

383

reported to be synthesized inside an alginate matrix (Dabiri et al., 2016).

f

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In general terms, the greater the precursor concentration the faster the nucleation rate

385

and crystal growth, so more well defined structures are formed, as for example the flower-

386

like type obtained by synthesizing brushite by the high internal phase emulsion method

387

(Lim et al., 2009).

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In the present work, larger particles were obtained from non heat-treated L-AEWB (170-

389

230 m diameter) than from heat-treated L-AEWB (110-170 m), the latter agglomerated

390

each other. This was probably due to the presence of organic molecules on surface, such as

391

carbohydrates contained in the wheat bran extracts.

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Although no N was detected in particles (Table 2), is assumed that proteins are present

393

in low quantities according to IR spectra (Fig. 4A) and SDS-PAGE (Fig. 7). Probably,

394

proteins are in the particle center where acted as nucleating sites, and so out of reach from

395

x-rays to be detected by EDS (Gazulla, Rodrigo, Blasco, Orduña, 2013). Luna-Valdez,

396

Balandrán-Quintana,

397

Madera-Santana et al. (2019) reported formation of spheroidal biopolymer particles,

398

average diameter 0.270 m, after heat treatment of L-AEWB. However, in the present work

399

brushite was produced even if L-AEWB was not subjected to heat treatment, the difference

400

just being larger particles. In view of this, thermal transition of proteins could not be the

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Azamar-Barrios,

Ramos

Clamont-Montfort,

Mendoza-Wilson,

17 Page 17 of 36

401

driving force to expose nucleating sites but rather establishes a boundary to crystal growth

402

through a yet unidentified way. More work is currently carried out in our lab to elucidate

403

mechanisms. Granular CaP particles have good biological properties, low cost, and broad availability,

405

so are the most used as bone graft substitutes. Among granular CaP particles, spherical ones

406

are of even greater interest because they improve handling properties of putties into which

407

are incorporated, such as injectability. However, making spherical CaP particles is not an

408

easy task (Bohner et al., 2013). There are several template methods for producing spherical

409

brushite particles. By reverse microemulsion is possible to obtain stable nanocrystalline

410

particles with average diameter in the range 23-87 nm. Precursor concentration and

411

temperature are key to tune particle size and morphology: the larger the precursor

412

concentration the larger the particle size, whereas spherical nanoparticles are obtained at 60

413

°C, in contrast to nanoflakes at 6 °C (Singh et al., 2010). Cement past emulsion, although

414

laborious, is a succesful method to fabricate clinically relevant spheres of brushite (200-

415

1,000 m) at low temperature, with a microstructure consisting of irregular shaped small

416

crystals in a size range of 0.7-7 m (Moseke et al., 2012). In the present work, spheroidal

417

brushite particles within approximately the same relevant size range were spontaneously

418

formed through a simple process.

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3.7. Elemental composition

421

Elemental composition of mineral phases is shown in Table 2 and is the average of 3

422

different examined areas. Carbon presence is explained at initial pH 5, where weddellite

423

(Ca(C2O4)∙2(H2O)) and brushite were jointly produced (Fig. S6), as well as at pH 11, where

18 Page 18 of 36

424

a mix of organic material was recovered, but not at pH 6.27 and 8, where pure brushite was

425

found. The origin of this C in the latter cases is probably the polymer that contains the

426

adhesive tape used in EDS analysis. Ca/P ratios around 1.0 at initial pH 6.27 and 8 are

427

characteristic of brushite, in concordance with results of XRD analysis. Those Ca/P ratios

428

corresponding to initial pH 11 are just coincidence as no crystalline phase was found under

429

this condition. Is interesting to note that P in brushite particles comes from aqueous extracts since no

431

phosphate preparation was used in the process, unlike Ca that was intentionally added.

432

Mineral reserve of wheat bran is in the globoids, spherical bodies immersed in the

433

innermost layer of bran, known as aleurone. Globoids contain P, K and Mg, but not Ca

434

(Luna-Valdez et al., 2017). In the globoids, P forms part of phytates, cationic salts of

435

inositol-6-phosphate (IP6), the latter known as phytic acid (Gupta, Gangoliya, Singh,

436

2015). Each of the six phosphate groups in the IP6 molecule can potentially to carry one

437

negative charge, depending on pH, and so is able to react with cations like Ca2+ (Nissar,

438

Ahad, Hussain, Naik, 2017). Since IP6 is usually extracted with 0.1 N HCl, and in the

439

present work a simple aqueous extraction step was performed, there would hardly be free

440

IP6 in the L-AEWB; however, it could be forming complexes with proteins or

441

polysaccharides. Biomineralization took place at pH near from the physiological one, so

442

phosphate groups from IP6 could be partially ionized and in a spatial relationship required

443

to attract calcium ions and form nuclei for the growth of brushite crystals. Nevertheless,

444

this remains to be elucidated.

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445

Brushite yield was determined through biomineralization at greater scale. For this, 100

446

mL AEWB 10% w/v (obtained by the straight process), were added with 10 mL of 0.75 M

447

CaCl2. Biomineralization was conducted by triplicate during 10 d, initial pH 6.23, 6±2 °C. 19 Page 19 of 36

448

Brushite particles was recovered as explained in materials and methods, and characterized.

449

Stereoscopical and SEM images showed particles identical to those found in the

450

microsystem. Elemental composition, FTIR spectrum, and XRD diffractograms were all

451

indicative of brushite phase (Fig S9). Brushite yield was 6.5±0.9 g by 100 g of AEWB.

452

3.8. Electrophoresis

454

In the electrophoresis gel, 3 bands of 5, 40, and 55 kDa were revealed (Fig. 7), which

455

support that discussed in Section 3.4 regarding posible presence of protein. In aqueous

456

extracts of wheat bran, a profile of molecular masses between 5 and 97 kDa has previously

457

been identified (Chaquilla-Quilca, Balandran-Quintana, Azamar-Barrios, Ramos-Clamont

458

Montfort, Mendoza-Wilson, Mercado-Ruiz et al., 2016; Chaquilla-Quilca, Balandrán-

459

Quintana, Huerta-Ocampo, Ramos-Clamont Montfort, Luna-Valdez, 2018). If, as

460

suspected, proteins act as nucleating sites, this would indicate that among these contained

461

in the organic matrix, only those of 5, 40, or 55 kDa have the necessary characteristics to

462

induce formation of brushite.

463 464

4. Conclusions

465

After the addition of calcium ions to aqueous wheat bran extracts, the latter lyophilized and

466

reconstituted in water, spheroidal brushite particles with diameters 110-200 m are

467

precipitated. Brushite is synthesized in a final pH range of 5.3-6.3 during an incubation

468

period of 10-15 days at 6±2 °C. The internal microstructure of the brushite particles

469

consists of elongated plates, approx. 20 nm thick, 200 nm wide and several microns in

470

length, which are stacked and aligned radially from the center of the particles to the outer

471

surface. Plates edges look flat, which gives to particles the feature of having an almost

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20 Page 20 of 36

472

smooth surface. It was demonstrated that aqueous wheat bran extracts act as scaffolds for

473

brushite biomineralization.

474 475

Funding: This work was supported by CONACYT, México [grant number A1-S-40197].

476

Acknowledgements

478

To CONACYT for the scholarship granted to author Zavala-Corrales for MSc studies. The

479

SEM, EDS, FTIR, and XRD analyzes were performed at the National Laboratory of Nano

480

and Biomaterials, Cinvestav-IPN-Mérida; financed by the projects FOMIX-Yucatán 2008-

481

108160, CONACYT LAB-2009-01-123913, 292692, 294643, 188345 and 204822. Thanks

482

to Dra. Patricia Quintana Owen, for enabling the access to LANNBIO; to M.C. Dora

483

Huerta Quintanilla, for technical support in SEM-EDX analysis; to M.C. Daniel Aguilar

484

Treviño, for his technical support in obtaining diffractograms, and to J.E. Corona, for

485

corrective maintenance of diffractometer.

486

Conflict of interest

487

Authors declare no conflict of interest.

488

Figure legends

489 490 491

Scheme 1. Experimental strategy for biomineralization in solution, using aqueous extracts of wheat bran (AEWB) as organic scaffolds. NL-AEWB = non lyophilized AEWB; LAEWB = lyophilized AEWB.

492 493 494

Figure 1. Precipitate yield as a function of incubation time and CaCl2 concentration, during biomineralization in solution of 10% w/v L-AEWB, with no heat treatment.

495 496 497 498

Figure 2. Morphology of representative particles obtained by biomineralization in solution, using aqueous extracts of wheat bran (AEWB). (A) Stereoscopical view. (B) SEM microphotography

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21 Page 21 of 36

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Figure 4. (A) IR spectra of precipitated particles after bio-mineralization of L-AEWB in solution (10% w/v), which was subjected or not to a heat pre-treatment at 68.5 °C for 3 h. (B) X-ray diffractograms of mineral phases after bio-mineralization of L-AEWB in solution (10% w/v), initial pH 6.27, which was subjected or not to a heat pre-treatment at 68.5 °C for 3 h. Incubation was 10 d at 6±2 °C after addition of 0.75 M CaCl2. HT: with heat pretreatment; NHT: with no heat pre-treatment.

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Figure 5. SEM microphotographs of mineral phases resulting from biomineralization of LAEWB (10% w/v), obtained by (A) the straight process or (B) the batch process. Biomineralization was performed 10 d at 6±2 °C after adding 0.75 M CaCl2, initial pH 8. L-AEWB was subjected (HT) or not (NHT) to heat pre-treatment before adding CaCl2. Each row shows different magnifications of images coming from a same treatment.

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Figure 6. SEM microphotographs of mineral phases resulting from biomineralization of 10% w/v L-AEWB, obtained by the straight process. Biomineralization was performed with 0.75 M CaCl2 at different initial pH, during a period of 10 d at 6±2 °C. L-AEWB was not subjected to heat treatment before adding CaCl2. Magnifications of each image are shown to its right.

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Figure 7. SDS-PAGE gels of proteins contained in brushite particles, synthesized in aqueous solution of L-AEWB (10% w/v). Electrophoresis was performed under reducing conditions, 12% gel, 14 mA. Lanes 1 and 2, broad- and low-MW markers, respectively. Lanes 3 and 4, replicated samples.

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Figure 3. Appearance of L-AEWB preps (10% w/v) at (A) zero time, and (B) after 10 d of biomineralization at initial pH 5, 6.27, or 8. Incubation temperature was 6±2 °C. Zero time is that just after addition of CaCl2.

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529

Conflict of interest

530

Authors declare no conflict of interest.

531

References

532 533 534 535 536

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Barth, A., Infrared spectroscopy of proteins, Biochimica et Biophysica Acta (BBA) Bioenergetics, 1767 (2007) 1073-1101. https://doi.org/10.1016/j.bbabio.2007.06.004 Bhojani, A., Jethva, H., Joshi, M., in: Growth inhibition study of urinary type brushite crystal using potassium dihydrogen citrate solution, DAE Solid State Physics Symposium 2018. Hisar, India. AIP Conference Proceedings, 2019; AIP Publishing. Bohner, M., Tadier, S., van Garderen, N., de Gasparo, A., Dobelin, N., Baroud, G., Synthesis of spherical calcium phosphate particles for dental and orthopedic applications, Biomatter, 3 (2013) https://doi.org/10.4161/biom.25103 Boistelle, R., Lopez-Valero, I., Growth units and nucleation: The case of calcium phosphates, J. Cryst. Growth, 102 (1990) 609-617. https://doi.org/10.1016/00220248(90)90420-P Boontaganon, P., Total, soluble and insoluble oxalate content of bran and bran products, J. Food. Agric. Environ., 7 (2009) 204-206. Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72 (1976) 248-254. https://doi.org/10.1016/0003-2697(76)90527-3 Brouns, F., Hemery, Y., Price, R., Mateo Anson, N., Wheat aleurone: Separation, composition, health aspects, and potential food use, Crit. Rev. Food Sci. Nutr., 52 (2012) 553-68. https://doi.org/10.1080/10408398.2011.589540 Brundavanam, S., Poinern, E., Fawcett, D., Growth of flower-like brushite structures on magnesium substrates and their subsequent low temperature transformation to hydroxyapatite, Am. J. Biomed. Eng., 4 (2014) 79-87. https://doi.org/10.5923/j.ajbe.20140404.02 Caddarao, P.S., Garcia-Segura, S., Ballesteros, F.C., Huang, Y.-H., Lu, M.-C., Phosphorous recovery by means of fluidized bed homogeneous crystallization of calcium phosphate. Influence of operational variables and electrolytes on brushite homogeneous crystallization, J. Taiwan Inst. Chem. Eng., 83 (2018) 124-132. https://doi.org/10.1016/j.jtice.2017.12.009 Campas-Ríos, M.d.J., Mercado-Ruiz, J.N., Valdéz-Covarrubias, M.A., Islas-Rubio, A.R., Mendoza-Wilson, A.M., Balandrán-Quintana, R.R., Hydrolysates from wheat bran albumin as color-adding agents and inhibitors of apple polyphenol oxidase, J. Food Biochem., 36 (2012) 470-478. https://doi.org/10.1111/j.1745-4514.2011.00553.x Chaquilla-Quilca, G., Balandran-Quintana, R.R., Azamar-Barrios, J.A., Ramos-Clamont Montfort, G., Mendoza-Wilson, A.M., Mercado-Ruiz, J.N., Madera-Santana, T.J., Lopez-Franco, Y.L., Luna-Valdez, J.G., Synthesis of tubular nanostructures from wheat bran albumins during proteolysis with v8 protease in the presence of calcium ions, Food Chem., 200 (2016) 16-23. https://doi.org/10.1016/j.foodchem.2016.01.005 Chaquilla-Quilca, G., Balandrán-Quintana, R.R., Huerta-Ocampo, J.Á., Ramos-Clamont Montfort, G., Luna-Valdez, J.G., Identification of proteins contained in aqueous extracts of wheat bran through a proteomic approach, J, Cereal Sci., 80 (2018) 3136. https://doi.org/10.1016/j.jcs.2018.01.005 Dabiri, S.M.H., Lagazzo, A., Barberis, F., Farokhi, M., Finochio, E., Pastorino, L., Characterization of alginate-brushite in-situ hydrogel composites, Mater. Sci. Eng. C, 67 (2016) 502-510. https://doi.org/10.1016/j.msec.2016.04.104

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Inoue, M., Hirasawa, I., The relationship between crystal morphology and XRD peak intensity on CaSO4·2H2O, J. Cryst. Growth, 380 (2013) 169-175. https://doi.org/10.1016/j.jcrysgro.2013.06.017 Izatulina, A.R., Punin, Y.O., in: Formation of calcium oxalates in the human body, Berlin, Heidelberg, 2012; Springer Berlin Heidelberg: Berlin, Heidelberg, 2012; pp 345349. Lim, H., Kassim, A., Huang, N., Khiewc, P., Chiu, W., Three-dimensional flower-like brushite crystals prepared from high internal phase emulsion for drug delivery application, Colloids Surf. A, 345 (2009) 211-218. https://doi.org/10.1016/j.colsurfa.2009.05.008 Luna-Valdez, J.G., Balandrán-Quintana, R.R., Azamar-Barrios, J.A., Ramos ClamontMontfort, G., Mendoza-Wilson, A.M., Madera-Santana, T.J., Rascón-Chu, A., Chaquilla-Quilca, G., Assembly of biopolymer particles after thermal conditioning of wheat bran proteins contained in a 21–43 kda size exclusion chromatography fraction, Food Hydrocoll., 94 (2019) 144-151. https://doi.org/10.1016/j.foodhyd.2019.03.003 Luna-Valdez, J.G., Balandrán-Quintana, R.R., Azamar-Barrios, J.A., Ramos ClamontMontfort, G., Mendoza-Wilson, A.M., Mercado-Ruiz, J.N., Madera-Santana, T.J., Rascon-Chu, A., Chaquilla-Quilca, G., Structural and physicochemical characterization of nanoparticles synthesized from an aqueous extract of wheat bran by a cold-set gelation/desolvation approach, Food Hydrocoll., 62 (2017) 165-173. https://doi.org/10.1016/j.foodhyd.2016.07.034 Lundager Madsen, H.E., Influence of foreign metal ions on crystal growth and morphology of brushite (CaHPO4·2H2O) and its transformation to octacalcium phosphate and apatite, J. Cryst. Growth, 310 (2008) 2602-2612. https://doi.org/10.1016/j.jcrysgro.2008.01.047

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Mandel, S., Tas, A.C., Brushite (CaHPO4·2H2O) to octacalcium phosphate (Ca8(HPO4)2(PO4)4·5H2O) transformation in DMEM solutions at 36.5 °C, Mater. Sci. Eng. C, 30 (2010) 245-254. https://doi.org/10.1016/j.msec.2009.10.009 Manissorn, J., Fong-Ngern, K., Peerapen, P., Thongboonkerd, V., Systematic evaluation for effects of urine ph on calcium oxalate crystallization, crystal-cell adhesion and internalization into renal tubular cells, Sci. Rep., 7 (2017) 1798-1798. https://doi.org/10.1038/s41598-017-01953-4 Marshall, R.W., Nancollas, G.H., Kinetics of crystal growth of dicalcium phosphate dihydrate, J. Phys. Chem., 73 (1969) 3838-3844. https://doi.org/10.1021/j100845a045 Mekmene, O., Quillard, S., Rouillon, T., Bouler, J.-M., Piot, M., Gaucheron, F., Effects of ph and ca/p molar ratio on the quantity and crystalline structure of calcium phosphates obtained from aqueous solutions, Dairy Sci. Technol., 89 (2009) 301316. https://doi.org/10.1051/dst/2009019 Miller, M.A., Kendall, M.R., Jain, M.K., Larson, P.R., Madden, A.S., Tas, A.C., Testing of brushite (CaHPO4·2H2O) in synthetic biomineralization solutions and in situ crystallization of brushite micro-granules, J. Am. Ceram. Soc., 95 (2012) 21782188. https://doi.org/10.1111/j.1551-2916.2012.05186.x Moseke, C., Bayer, C., Vorndran, E., Barralet, J.E., Groll, J., Gbureck, U., Low temperature fabrication of spherical brushite granules by cement paste emulsion, J. Mater. Sci. Mater. Med., 23 (2012) 2631-7. https://doi.org/10.1007/s10856-012-4740-1 Nissar, J., Ahad, T., Hussain, S., Naik, H.R., A review phytic acid: As antinutrient or nutraceutical, J. Pharmacogn. Phytochem., 6 (2017) 1554-1560. https://bit.ly/39fuc22 Rubini, K., Boanini, E., Bigi, A., Role of aspartic and polyaspartic acid on the synthesis and hydrolysis of brushite, J. Funct. Biomater., 10 (2019) 11. https://doi.org/10.3390/jfb10010011 Singh, S., Singh, V., Aggarwal, S., Mandal, U.K., Synthesis of brushite nanoparticles at different temperatures, Chem. Pap., 64 (2010) 491-498. https://doi.org/10.2478/s11696-010-0032-8 Suryawanshi, V.B., Chaudhari, R.T., Growth and characterization of agar gel grown brushite crystals, Indian J. Mater. Sci., 2014 (2014) 6. https://doi.org/10.1155/2014/189839 Tamimi, F., Sheikh, Z., Barralet, J., Dicalcium phosphate cements: Brushite and monetite, Acta Biomater., 8 (2012) 474-87. https://doi.org/10.1016/j.actbio.2011.08.005 Tas, A.C., Bhaduri, S.B., Chemical processing of CaHPO4·2H2O, J. Am. Ceram. Soc., 87 (2004) 2195-2200. https://doi.org/10.1111/j.1151-2916.2004.tb07490.x Toshima, T., Hamai, R., Tafu, M., Takemura, Y., Fujita, S., Chohji, T., Tanda, S., Li, S., Qin, G.W., Morphology control of brushite prepared by aqueous solution synthesis, J. Asian Ceram. Soc., 2 (2014) 52-56. https://doi.org/10.1016/j.jascer.2014.01.004 Trpkovska, M., Šoptrajanov, B., Malkov, P., FTIR reinvestigation of the spectra of synthetic brushite and its partially deuterated analogues, J. Mol. Struct., 480-481 (1999) 661-666. https://doi.org/10.1016/S0022-2860(98)00923-5 Zhang, J., Wang, L., Putnis, C.V., Underlying role of brushite in pathological mineralization of hydroxyapatite, J. Phys. Chem. B, 123 (2019) 2874-2881. https://doi.org/10.1021/acs.jpcb.9b00728

Jo u

628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674

25 Page 25 of 36

675 676 677 678 Table 1. Recorded pH during 10 d biomineralization of L-AEWB (10% w/v) with initial pH 5, 6.27, or 8. Biomineralization started by adding 0.75 M CaCl2. Incubation was done at 6.0±2.0 °C. Initial pH (prior to CaCl2 addition) x

Time (d)

5.0

a

6.27

a

8.0

a

4.8±0.06

3

4.9±0.06

4

4.8±0.06

5

5.0±0.06

6

4.9±0.06

7

4.9±0.06

8

5.0±0.06

9

5.0±0.06

10

5.0±0.06

a

5.5±0.06

a

5.4±0.06

a

5.4±0.06

a

5.2±0.06

a

5.3±0.06

a a a

b b b b

5.3±0.06 5.2±0.06 5.1±0.06

a

5.1±0.06

a

5.3±0.06

b b b b b b

6.3±0.06 6.2±0.06 6.2±0.06 6.2±0.06 6.2±0.06 6.3±0.06 6.2±0.06 6.3±0.06 6.2±0.06 6.3±0.06 6.3±0.06

c c c c c c c c c c c

rn

x

oo

2

b

pr

4.9±0.06

5.5±0.06

e-

1

a

Pr

4.7±0.06

al

0

f

Recorded pH as function of time

pH at zero time is that registered just after CaCl2 was added. Recorded pH is the average of 3 reps

679 680 681 682

Jo u

± standard error. Different superscript letters in a same pH column indicate significant differences at p≤0.05.

26 Page 26 of 36

Table 2. Elemental composition of precipitated particles after 10 d biomineralization of LAEWB 10% w/v, with 0.75 M CaCl2, in a range of initial pH. Initial pH 5

6.27

8

11

C

Weight Atomic % % 13.37 20.69

Weight Atomic % % 10.61 10.44

Weight Atomic % % 7.91 12.57

Weight Atomic % % 17.10 26.97

O

53.65

62.31

57.27

66.62

57.78

43.47

51.46

P

12.32

7.39

14.85

8.92

15.35

9.46

15.17

9.28

Ca

19.89

9.22

17.27

8.02

18.95

902

21.02

9.93

Mg

--

--

--

--

Cl

0.41

0.22

--

--

K

0.37

0.17

--

--

Ca/P

1.61

1.25

1.16

f

oo

e-

pr

2.07

--

--

--

--

--

--

0.59

0.28

0.9

1.23

0.95

1.39

1.07

Jo u

686

2.66

rn

684 685

--

al

683

68.94

--

Pr

Element

27 Page 27 of 36

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