WO2010058049A1 - Preparation of biocompatible materials from waste from the process for manufacturing beer and uses thereof - Google Patents

Abstract

The present invention refers to the process of obtainment of biocompatible material comprising phosphates and silicates from waste from the agri-food industry, more specifically from drying and heat treatment of wet brewer’s grains, a residue from the manufacture of beer. The invention furthermore relates to the use of these biocompatible materials in osseous tissue engineering.

Description

 PREPARATION OF BIOCOMPATIBLE MATERIALS FROM WASTE FROM THE BEER MANUFACTURING PROCESS AND ITS USES

TECHNICAL SECTOR The present invention relates to a method of obtaining biocompatible material from the drying and heat treatment of beer bagasse, as well as its use in bone tissue engineering. Therefore, from the point of view of the process, the present invention falls within the sector of the synthesis and preparation of new materials. Regarding its applications or uses, the invention falls within the health sector, and more specifically in bone tissue implants.

STATE OF THE TECHNIQUE

At present, worldwide, biomaterial engineering has aroused great interest, becoming of great economic importance, as a viable option to replace tissues and organs, given the progressive increase in

The average age of the population. The current development of tissue engineering is based both on the development of cultures of more or less pluripotential cell lines, inducing and regulating factors of cell differentiation, and on the development of matrices with support and structure capacity. Specifically in the development of bone regeneration, multiple models of osteoconductive characteristics, rich in phosphocalcic contents, have been designed.

Usually, a part of them comes from the treatment of animal structures from which their protein components are totally or partially removed, conserving the mineralized phase and another comes from the calcium phosphates by means of synthesis processes.

Numerous surgeries need to restore tissues, often in the presence of insufficient amounts of natural material and therefore regeneration procedures are a primary part of these therapies. Biomaterials used must have the appropriate characteristics of composition, mechanical strength, porosity, degradation rate in the appropriate biological solutions, material transport properties and exchange of nutrients and waste products, using polymers, proteins and inorganic materials (alumina, zirconia, hydroxyapatite, calcium phosphates and bioactive glasses), mainly. [Matthew B. Murphy and Antonios G. Mikos (2007), "Principies of Tissue Engineering" (Third Edition), 309-321. Chapter Twenty-Two - Polymer scaffold fabrication] [SJ Kalita, A. Bhardwaj and HA Bhatt (2007), Nanocrystalline calcium phosphate ceramics in biomedical engineering, Materials Science and Engineering: C, 27, 441-449]

Usually the solids used are synthetic (AL Oliveira, PB Malafaya, RL Reis (2003), Sodium silicate gel as a precursor for the in vitro nucleation and growth of a bone-like apatite coating in compact and porous polymeric structures, Biomaterials, 24, 2575-2584), (AG Dias, IR Gibson, JD Santos, MA Lopes (2007), Physicochemical degradation studies of calcium phosphate glass ceramic in the CaO-P2O5-MgO-TiO2 system, Acta Biomaterialia, 3, 263-269) or of animal origin, although recently the diseases related to the bovine population have raised the fear among human beings in implanting materials with such origin, being more recommended the use of materials of non-animal origin. Synthetic materials, on the other hand, are often obtained by complicated synthesis of a large number of synthetic steps, using toxic reagents (eg benzoyl peroxide, benzene, anilines), with calcinations at very high temperatures close to 1500 ⁰ C , to produce bioceramic materials, to which silicon is added to finish, by hydrolysis of TEOS, with a final sintering step at more than 1100 ⁰ C. [MB Nair, SS Babu, HK Varma, A. John (2008) . A triphasic ceramic-coated porous hydroxyapatite for tissue engineering application, Acta Biomaterialia, 4, 173-181]

The present work is based on obtaining biocompatible solids of added value using as raw material by-products of agricultural origin, carrying out processes consistent with sustainable development, that is, avoiding the use of toxic substances or procedures. The structure of the Final materials can be designed by changing the variables of the preparation process, so that they can be used in cell growth, according to their final textural and crystalline characteristics.

BRIEF DESCRIPTION OF THE INVENTION

The present invention is based on three fundamental aspects: 1) the obtaining of materials comprising phosphates and silicates, by drying and heat treating the bagasse (previous residues of the beer manufacturing process), 2) the material obtained by this procedure 3) its use in bone tissue engineering, as well as its use as a catalyst.

DETAILED DESCRIPTION

The present invention is based on a process, developed by the inventors, to obtain biocompatible materials comprising phosphates and silicates from bagasse residues of beer. The materials obtained, given their similarity to the bone mineral phase, are suitable for bone tissue engineering.

Therefore, one aspect of the present invention is the process of obtaining materials comprising phosphates and silicates, hereinafter the method of the invention, by drying and heat treating the bagasse of beer.

In this invention beer bagasse is defined as the byproduct of

The beer industry resulting from the pressing and filtering of the must obtained from the malted barley grain, after its saccharification. The bagasse of beer has a large amount of liquid, due to the processing of the original materials, and a dry matter content of 20-25%.

The large amount of liquid in the beer bagasse makes its separation from the solid necessary. Said solid will be treated, subsequently, by conventional heating, varying temperatures and times to control the textural characteristics of the final material, also depending on the chemical composition and the type and quantity of pores of the material.

A preferred aspect of the present invention is the process of the invention that is developed through the steps of: i) Drying of beer bagasse by heating from room temperature to 100-200 ⁰ C with a temperature ramp between 1 and 10 ° C / min and maintaining the final temperature for at least two hours to stop the fermentation of the original solid. ii) Treatment of the resulting solid in i) from room temperature to a temperature within the range of 350-1200 ⁰ C, maintaining the final temperature for more than 1 hour

A more preferred aspect of the present invention is the process of the invention in which the final temperature of stage i) is 15O ⁰ C.

Another more preferred aspect of the present invention is the process of the invention in which the final temperature of stage ii) is 85O ⁰ C, giving rise to a material comprising phosphorus, silicon, calcium and magnesium as main elements, in addition to lower amounts of sodium and potassium. By different characterization techniques (see example 1) it has been shown that the material obtained by the process of the invention, using this final temperature of 85O ⁰ C, can be used in bone tissue engineering.

Another more preferred aspect of the present invention is the process of the invention in which the heating ramp used in i) is 5 ° C / min.

Another more preferred aspect of the present invention is the process of the invention in which the final temperature in stage i) is maintained for 4 hours.

Another more preferred aspect of the present invention is the process of the invention in which the final temperature in stage ii) is maintained for 2 hours. Another more preferred aspect of the present invention is the process of the invention in which the final temperature of stage ii) is 35O ⁰ C, giving rise to a material comprising carbon, phosphorus, silicon, calcium and magnesium, in addition to amounts lower sodium and potassium. By different characterization techniques (see example 2) it has been shown that the material obtained by the process of the invention, using this final temperature of 35O ⁰ C, can be used as a catalyst, absorber or semiconductor.

Another aspect of the present invention is the material obtained by the process of the invention, hereinafter material of the invention. The observation of the material of the invention with X-ray diffraction determines a crystalline structure corresponding to calcium and magnesium phosphates and silicates. Textural analysis of the material of the invention indicates the presence of macropores above 100 microns and a specific area of around 8m ² / g. The results of chemical analysis and microwave analysis confirm the presence of phosphorus, silicon, calcium and magnesium as the main elements of the material of the invention, in addition to lower amounts of sodium and potassium.

Another preferred aspect of the invention is the material of the invention characterized in that it mostly comprises phosphorus, silicon, calcium and magnesium, in addition to lower amounts of sodium and potassium.

Given its similarity with the mineral phase of the bone, the material of the invention is suitable for the engineering of bone tissues and also its content in ions present in the physiological environment (sodium, calcium, magnesium, potassium) makes it highly biocompatible. The presence of phosphorus, silicon and magnesium is beneficial for tissue engineering, since phosphates are usually more soluble in biological fluids than hydroxyapatite. The presence of silicon is used to modify the dissolution capacity of the biomaterial, having found a greater dissolution of silicon ions and faster reabsorption in silica-calcium phosphate composites than in materials without silica. Also, the presence of biologically active cations such as magnesium is a contributing factor in the properties of this type of biomaterials, since it influences the process of the subsequent biomineralization of the solid. Bioreabsorbable materials are important for tissue regeneration, since they do not require a second operation to remove the implant.

Therefore, another aspect of the present invention is the use of the material of the invention in bone tissue engineering

Thus, the material of the invention, given its biocompatibility and its textural characteristics with pore diameters of several hundred microns, which can be modified by procedures designed in this regard, is useful for the growth of fat stem cells. This is the first time that a renewable material, given its origin of agricultural waste, after the necessary modifications, is used for the cultivation of stem cells from fat. Natural coral-based materials have been used for similar purposes, although their sustainability is doubtful, since coral materials cannot be considered renewable and their presence is very important for the balance of the environment where they develop.

The plant origin of the material of the invention, from residues of the agricultural industry, prevents the possible spread of diseases that can be transmitted through the bone mass of animals. In addition, the material of the invention contains silicon in its composition, so it is not necessary to add it to modify the hardness and biodegradability of the final biomaterials, with the consequent economic benefit.

Another aspect of the present invention is the use of the material of the invention as a catalyst.

EXAMPLES OF EMBODIMENT OF THE INVENTION

EXAMPLE 1 - Preparation of biocompatible material from beer bagasse and characterization to verify its application in bone tissue engineering. The beer bagasse obtained directly from the MAHOU factory in Alovera, Guadalajara, is dried by heating from room temperature to 15O ⁰ C with a temperature ramp of 5 ° C / min and maintaining the final temperature for four hours to stop the Ia fermentation of the original solid. Then, the resulting solid was heated from room temperature to a temperature of 850 ⁰ C, maintaining the final temperature for 2 hours.

The material obtained mainly comprises phosphorus, silicon, calcium and magnesium as the main elements, as well as lower amounts of sodium and potassium. The influence of its structural, surface, textural and biocompatibility characteristics on the growth of fat stem cells has been studied.

For the characterization of the crystalline phases of the material object of the patent, X-ray powder diffraction was used with an X ^' Pert Pro PANalytical Polycrystalline diffractometer. The X-ray diffraction diagram was carried out under standard conditions using Cu radiation Ka (λ = 1.54060 A). X-ray diffraction diagrams were made, with peaks between 22 and 25 ° and at 31, 33 and 34.5 2 Θ corresponding to tricalcium phosphate (PDF 09-0348) and calcium silicate at 29.5 ° 2 Θ (PDF 01 -1029). The peaks obtained for the material under study are comparable to those found in the literature for similar compounds used in bone tissue growth: β-Ca2P2θ7 (calcium pyrophosphate; 30.8 ° 2Θ) α-TCP (22.9 ° 2Θ). α-TCP and hydroxyapatite (31.6 ° 2Θ). β- Ca ₂ P ₂ θ ₇ (29.0 and 30.2 ° 2Θ). [D. Tadic and M. Epple, A thorough physicochemical characterization of 14 calcium phosphate-based bone substitution materials in comparison to natural bone, Biomaterials, 25, (2004) 987-994; A. Oyane, Y. Ishikawa, A. Yamazaki, Yu Sogo, K. Furukawa, T. Ushida, Atsuo Ito Reduction of surface roughness of a laminin-apatite composite coating via inhibitory effect of Mg ions on apatite crystal growth, Acta Biomaterialia, Volume 4, (2008) 1342-1348]. The porosities of the prepared materials were determined by nitrogen adsorption techniques in a Micromeritics ASAP 2010 device with N ₂ at 77 K and mercury porosimetry, in a Merons Pascal 140/240 mercury intrusion-extrusion equipment. For measurements of N ₂ adsorption samples evacuated to a process for 16 hours at 300 ⁰ C they were previously subjected to eliminate any kind that may be weakly adsorbed to the surface. The apparent specific surfaces (S _BET ) were obtained according to the BET method, assuming an area for the nitrogen molecule of 0.162 nm ² . Rouquerol, F., Rouquerol J., Sing, K .; Adsorption by powders and porous solids- principies, methodology and applications, (1999), Academic Press, London). Particle size and meso and macropore distributions were determined by mercury intrusion porosimetry, after drying the samples in an oven at 150 ° C for 16 hours. For these measurements, a mercury contact angle of 141 ° and a surface tension of 484 mNm ^"1 according to IUPAC recommendations are assumed as standard. The adsorption of nitrogen in the samples gives rise to type II isotherms.

Due to this characteristic, the apparent surface area was determined by the BET method in a relative pressure range p / p ° = 0.05-0.30.

The curve obtained in the measure of mercury intrusion porosimetry indicates particle sizes of several hundred microns. The porosity study reveals that the material has an open pore structure, with more than 80% of them between 100 and 300 microns in diameter. The bulk density of the material is 0.35 g / cc, indicating that it has a porosity of 86%. The skeletal density is 2.5 g / cc.

The porosity of similar solids, taken for comparison in bibliographic searches is usually in a range of 50 to 500 microns. The results of textural characterization confirm that depending on the parameters used for the design of the solids, they have the capacity to serve as scaffolds in cell growth, which are confirmed by analysis performed with scanning electron microscopy. With infrared spectroscopy (FTIR) bands are observed at 3570 cm ^"1 , which are attributed to hydroxyl groups and 1190-976 cm ^{" 1} and 660-520 cm ^"1 which are attributed to phosphate and / or silicate groups. Given the similarity of the vibrational modes of silicate and phosphate groups, it is difficult to distinguish their bands with this technique (Acta biomateralia 4 (2008) 173). Preparation and bioactivity evaluation of bone-like hydroxyapatite nanopowder, MH Fathia *, A. Hanifia, V. Mortazavi).

Carrying out cultures of stem cells from fat in this solid, it has been shown how these cells grow on their surface in a manner similar to what occurs in a hydroxyapatite of animal origin, commonly used in regenerative techniques and used as control. The absence of growth inhibition in the plate is another fact that corroborates its biocompatibility.

EXAMPLE 2 - Preparation of biocompatible material from beer bagasse and characterization to verify its application as a catalyst, substance absorber or semiconductor.

The beer bagasse obtained directly from the MAHOU factory in Alovera, Guadalajara, is dried by heating from room temperature to 15O ⁰ C with a temperature ramp of 5 ° C / min and maintaining the final temperature for four hours to stop the Ia fermentation of the original solid. Then, the resulting solid was heated from room temperature to a temperature of 350 ⁰ C, maintaining the final temperature for 2 hours.

The texture of this material was determined by the nitrogen adsorption-desorption technique in a Micromeritics ASAP 2010 equipment with N ₂ at 77 K, subjecting the solid previously to an evacuation process for 16 hours at 300 ⁰ C to eliminate any species that It could be weakly adsorbed on the surface. The apparent specific surfaces (S _BET ) were obtained according to the BET method, assuming an area for the nitrogen molecule of 0.162 nm ² [Rouquerol, F., Rouquerol J., Sing, K .; Adsorption by powders and porous solids- principies, methodology and applications, (1999), Academic Press, London]. For the characterization of the crystalline phases of the material object of the patent, X-ray powder diffraction was used with an X ^' Pert Pro PANalytical Polycrystalline diffractometer. The X-ray diffraction diagram was carried out under standard conditions using Cu radiation Ka (λ = 1.54060 A). The material obtained mainly comprises carbon, phosphorus, silicon, calcium and magnesium as the main elements, as well as lower amounts of sodium and potassium. The observation of this solid with X-ray diffraction allows its non-crystalline character to be determined and the textural analysis of this material indicates the presence of micropores and a specific surface area (S _BET ) of 500m ² / g.

Materials similar to this (carbon with phosphorus and silicon in its composition) have recently been used as semiconductors [Silicon-doped carbon semiconductor from rice husk char Materials Chemistry and Phvsics Volume 109, Issue 1. 15 May 2008, Pages 169-173 S. Maiti, P. Banerjee, S. Purakayastha and B. Ghosh], as catalysts [Catalytic performance of carbonaceous materials in the esterification of succinic acid Catalysis Communications 9 (2008) 1709-1714 JH Clark, V. Budarin, T. Dugmore, R Luque, DJ Macquarrie, V. Strelko] and as toxic substance adsorbents [Removal of trace organics from water using a pumped bed-membrane bioreactor with powdered activated carbon, CG Dosoretz and KW Boddeker, Journal of Membrane Science Volume 239, Issue 1 , 1 August 2004, Pages 81-90].

Claims

1. Procedure for obtaining materials comprising phosphates, silicates characterized in that it is made from drying and heat treatment of beer bagasse. 2. Method according to claim 1 characterized in that it is carried out through the steps of: i) drying of beer bagasse by heating from room temperature to 100-200 ⁰ C with a temperature ramp between 1 and 10 ° C / min and maintaining the final temperature for at least two hours to stop the fermentation of the original solid, ii) treatment of the resulting solid in i) from room temperature to a temperature within the range of 350-1200 ⁰ C, maintaining the final temperature for more than 1 hour. 3. Method according to claim 2 characterized in that the final temperature of stage i) is 15O ⁰ C. 4. Method according to claim 2 characterized in that the final temperature of stage ii) is 85O ⁰ C. 5. Method according to claim 2 characterized in that the heating ramp used in i) is 5 ° C / min. 6. Method according to claim 2 characterized in that the final temperature in stage i) is maintained for 4 hours. 7. Method according to claim 2 characterized in that the final temperature of stage ii) is 35O ⁰ C. 8. Method according to claim 2 characterized in that the final temperature in stage ii) is maintained for 2 hours. 9. Material obtained by the preceding claims. 10. Material obtained by claim 4 characterized in that it mostly comprises phosphorus, silicon, calcium and magnesium, in addition to lower amounts of sodium and potassium. 11. Material obtained by claim 7 characterized in that it mostly comprises carbon, phosphorus, silicon, calcium and magnesium, in addition to lower amounts of sodium and potassium 12. Use of the material obtained by claims 1-7, in bone tissue engineering. 13. Use of the material obtained by claims 1-7 as a catalyst. 14. Use of the material obtained by claims 1-7 as an adsorbent of toxic substances 15. Use of the material obtained by claims 1-7 as a semiconductor