Patent Application
20240158584
Gels are semisolid materials with a liquid phase entrapped in a three-dimensional network structure. Based on their textural and rheological properties, these materials provide many applications in the food, biomedical, pharmaceutical, cosmetics, wastewater remediation and agriculture industries. In recent years, natural polymer-based hydrogels (water-based gels) have been a topic of several studies due to their advantages (biocompatibility, biodegradability, accessibility, and renewability) over synthetic hydrogels.
More broadly, hydrogels are defined as a three-dimensional network of polymeric materials that can swell upon absorption of water while maintaining their structure by chemical or physical crosslinking of the constituent polymeric chains. Hydrogels can be categorized into different groups based on various criteria. These criteria include the source of the material (synthetic, natural, or hybrid), whether the material is crosslinked and how the crosslinking is achieved (chemically by covalent bonding; or physically by non-covalent bonding), biodegradability (biodegradable, non-biodegradable), polymeric structure (homopolymer, copolymer, interpenetrating network), and charge (neutral, ionic, amphoteric, and zwitterionic). Regarding source and polymeric structure, synthetic polymers, e.g., poly-2-hydroxyethyl methacrylate (pHEMA), polycaprolactone (PCL), polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), polylactic acid (PLA), polyacrylamide (PAM), polyethylene glycol (PEG), and their derivatives are the primary sources used for hydrogel fabrication on a commercial scale. See, for example, U. S. K. Madduma-Bandarage, S. V Madihally, Synthetic hydrogels: Synthesis, novel trends, and applications, J. Appl. Polym. Sci. 138 (2021) 50376. Soon after the development of the first synthetic hydrogel in 1960 by Wichterle and Lim using pHEMA (0. Wichterle, D. Lim, Hydrophilic gels for biological use, Nature. 185 (1960) 117-118), hydrogels were used to make soft contact lenses. In the ensuing years, the use of hydrogels has expanded significantly into fields including drug delivery, tissue engineering, wound dressing, wastewater remediation, agriculture, biosensors, cosmetics, and skincare products. Although synthetic hydrogels have desirable characteristics (e.g., high swelling and water-retention capacity, improved gel strength and firmness, more straightforward production processes, and extended shelf life), they are not without concerns. Among these concerns are their potential toxicity and lack of biocompatibility, along with environmental issues such as environmental degradability and waste disposal. Additionally, most synthetic hydrogels use polymers made from petrochemical feedstocks—making their prices fluctuate in unpredictable ways. Lastly, there has been a long-running desire to use abundantly available edible and affordable bioresources.
These concerns have drawn the attention of researchers toward renewable, bio-based hydrogels. See, for example, A. H. Karoyo, L. D. Wilson, A review on the design and hydration properties of natural polymer-based hydrogels, Materials (Basel). 14 (2021) 1095. The primary hydrogel-forming natural polymers include polypeptides (proteins) and polysaccharides; W. Wijaya, A. R. Patel, A. D. Setiowati, P. Van der Meeren, Functional colloids from proteins and polysaccharides for food applications, Trends Food Sci. Technol. 68 (2017) 56-69. Animal-based proteins such as collagen, gelatin, whey, elastin, and keratin and plant-based proteins like soy, zein, pea, faba bean, wheat gluten, and lentil are some examples of polypeptides used for hydrogel fabrication. (F. Zha, J. Rao, B. Chen, Plant-based food hydrogels: Constitutive characteristics, formation, and modulation, Curr. Opin. Colloid Interface Sci. 56 (2021) 101505; N. Ni, M.-J. Dumont, Protein-based hydrogels derived from industrial byproducts containing collagen, keratin, zein and soy, Waste and Biomass Valorization. 8 (2017) 285-300; and H. F. Darge, A. T. Andrgie, H.-C. Tsai, J.-Y. Lai, Polysaccharide and polypeptide based injectable thermo-sensitive hydrogels for local biomedical applications, Int. J. Biol. Macromol. 133 (2019) 545-563.) Pectin, agar, alginate, xanthan gum, starch, chitosan, chitin, hyaluronic acid, and cellulose derivatives are some of the polysaccharides utilized as building materials for producing natural hydrogels. See D. Pasqui, M. De Cagna, R. Barbucci, Polysaccharide-based hydrogels: the key role of water in affecting mechanical properties, Polymers (Basel). 4 (2012) 1517-1534; T. Zhu, J. Mao, Y. Cheng, H. Liu, L. Lv, M. Ge, S. Li, J. Huang, Z. Chen, H. Li, Recent progress of polysaccharide-based hydrogel interfaces for wound healing and tissue engineering, Adv. Mater. Interfaces. 6 (2019) 1900761; and A. Manzoor, A. H. Dar, V. K. Pandey, R. Shams, S. Khan, P. S. Panesar, J. F. Kennedy, U. Fayaz, S. A. Khan, Recent insights into polysaccharide-based hydrogels and their potential applications in food sector: A review, Int. J. Biol. Macromol. 213 (2022) 987-1006. Compared with homopolymers consisting of polysaccharides or proteins only, the combined biopolymer hydrogel often provides hierarchical microstructures of gels and diversified functional properties, depending on the intermolecular interactions between two biopolymers and their gelation mechanism. See X. Yang, A. Li, D. Li, Y. Guo, L. Sun, Applications of mixed polysaccharide-protein systems in fabricating multi-structures of binary food gels—A review, Trends Food Sci. Technol. 109 (2021) 197-210 and X. T. Le, L.-E. Rioux, S. L. Turgeon, Formation and functional properties of protein—polysaccharide electrostatic hydrogels in comparison to protein or polysaccharide hydrogels, Adv. Colloid Interface Sci. 239 (2017) 127-135.
There have been numerous reports on utilizing polysaccharide-protein mixtures as the building blocks of hydrogels. Some of the studied binary mixtures are konjac glucomannan/fish myofibrillar protein, low-methoxyl pectin (LMP)/pea protein, sorghum arabinoxylan/soy protein isolate (SPI), high-methoxyl sugar beet pectin/SPI, high methoxyl citrus pectin/whey protein isolate, and chitosan/gelatin, K-carrageenan/casein, dextrin/oat protein, and high-methoxyl sugar beet pectin/zein. See T. Zhang, S. Chen, X. Xu, X. Zhuang, Y. Chen, Y. Xue, C. Xue, N. Jiang, Effects of konjac glucomannan on physical properties and microstructure of fish myofibrillar protein gel: Phase behaviours involved, Food Hydrocoll. 134 (2023) 108034; D. Zhang, D. Chen, B. Patel, O. H. Campanella, Pectin as a natural agent for reinforcement of pea protein gel, Carbohydr. Polym. 298 (2022) 120038; J. Yan, L. Yin, Y. Qu, W. Yan, M. Zhang, J. Su, X. Jia, Effect of calcium ions concentration on the properties and microstructures of doubly induced sorghum arabinoxylan/soy protein isolate mixed gels, Food Hydrocoll. 133 (2022) 107997; E. G. Ates, E. B. Ozvural, M. H. Oztop, Understanding the role of d-Allulose and soy protein addition in pectin gels, J. Appl. Polym. Sci. 138 (2021) 49885; B. Ozel, 0. Aydin, M. H. Oztop, In vitro digestion of polysaccharide including whey protein isolate hydrogels, Carbohydr. Polym. 229 (2020) 115469; S. R. Derkach, Y. A. Kuchina, D. S. Kolotova, N. G. Voron'ko, Polyelectrolyte polysaccharide—gelatin complexes: Rheology and structure, Polymers (Basel). 12 (2020) 266; M. Tang, Y. Zhu, D. Li, B. Adhikari, L. Wang, Rheological, thermal and microstructural properties of casein/K-carrageenan mixed systems, Lwt. 113 (2019) 108296; T. V. N. Nieto, Y. Wang, L. Ozimek, L. Chen, Improved thermal gelation of oat protein with the formation of controlled phase-separated networks using dextrin and carrageenan polysaccharides, Food Res. Int. 82 (2016) 95-103; and S. Soltani, A. Madadlou, Two-step sequential cross-linking of sugar beet pectin for transforming zein nanoparticle-based Pickering emulsions to emulgels, Carbohydr. Polym. 136 (2016) 738-743, respectively. Despite the biocompatibility and biodegradability advantages of using biopolymers and the diversity of the sources of polysaccharides and proteins used in the literature studies, there is a drawback: the individual biopolymers from the animal or plant sources must be isolated. This not only significantly increases the cost of hydrogel production (due to the need for multi-stage downstream processing for isolation and purification), but also adversely influences the environment due the higher amounts of effluent discharged. These processing requirements significantly and adversely impact the economic feasibility and industrial scalability of hydrogel production from natural sources.
Soybeans are one of the most cultivated crops in the world. The seed coat of the soybean, i.e., soybean hull (SBH), is the primary byproduct of the soy industry. The SBH is separated from the bean during the dehulling step as a prerequisite for oil and protein extraction. Annual worldwide production of SBH runs from −18.0 to 28.7 million metric tons in 2020-2021. G. A. Bittencourt, L. P. de Souza Vandenberghe, K. Valladares-Diestra, L. W. Herrmann, A. F. M. de Mello, Z. S. Vasquez, S. G. Karp, C. R. Soccol, Soybean hulls as carbohydrate feedstock for medium to high-value biomolecule production in biorefineries: A review, Bioresour. Technol. 339 (2021) 125594. Although SBH is generated in enormous quantities, the vast majority of it is used in low-value applications, mostly as animal feed. A significant percentage of SBH is simply incinerated or landfilled, which raises additional environmental and health issues.
Disclosed herein is a method to make hydrogels from pectin- and protein-containing cellulosic biomass in a single step. In the method, the biomass is treated with an aqueous acidic solution for a time, at a temperature, and at a pH sufficient to yield a mixture containing biomass solids, hydrogels. The acid within the aqueous acidic solution may be added exogenously or may be formed in situ. In the preferred version, the novel approach produces three products without generating effluent:
1. A gel fraction: The pectin- and protein-containing hydrogel formed by the method is amphiphilic. It has several important utilities, including as a rheology modifier (i.e., an additive used to adjust the viscosity and non-Newtonian behavior of substances with complex microstructures), as an encapsulant for nutrients, bioactive materials, pharmaceutically active materials, and the like, and as an emulsion stabilizer in food, cosmetics, pharmaceuticals, paints, coatings, and adhesives
2. A solids fraction: The solids formed by the method are cellulose-rich and are useful for making a wide range of cellulose-based products, including but not limited to, microcrystalline cellulose, nanocrystalline cellulose, cellulose ethers, cellulose acetates, cellulose nitrates, and carboxymethyl cellulose.
3. A liquid fraction: The dissolved biomass mainly contains products from the degradation of pectin, protein, hemicellulose, and cellulose. It is useful in a wide range of applications, including but not limited to, bio-based films and coatings.
Unlike the methods reported in the literature, the present hydrogel fabrication method does not need to isolate a single component (polysaccharide or protein) from the lignocellulosic biomass. Separating individual components requires extensive, costly, and environmentally harmful downstream processing. The yield and properties of the hydrogel formed by the method are adjustable by varying the processing conditions of the feedstock (i.e., pH, time, and temperature).
Thus, disclosed herein is a method of making a hydrogel. In a first version, the method comprises treating a cellulosic biomass comprising pectin and protein with an aqueous acidic solution at a pH, at a temperature, and for a time sufficient to cause formation of at least a gel fraction comprising the hydrogel.
Another version of the method comprises treating a cellulosic biomass comprising pectin and protein with an aqueous acidic solution at a pH, at a temperature, and for a time sufficient to cause formation of at least a gel fraction comprising the hydrogel. The treated cellulosic biomass is then blended to yield a mixture. The mixture is then optionally cooled to, e.g., from about 10° C. to about 0° C., most preferably about 4° C., to yield a cooled mixture. The cooled mixture is then centrifuged to concentrate the gel fraction.
A specific version of the method comprises treating a cellulosic biomass comprising pectin and protein with an aqueous acidic solution at a pH, at a temperature, and for a time sufficient to cause formation of at least a gel fraction comprising the hydrogel. Then blending the treated cellulosic biomass to yield a mixture and cooling the mixture to a temperature of about 10° C. to about 0° C. to yield a cooled mixture. Lastly, the cooled mixture is centrifuged to concentrate the gel fraction.
The preferred pH of the treatment step is from about 1 to about 6, more preferably from about 1.8 to about 5.8. The preferred temperature of the treatment step is from about 30° C. to about 150° C., or from about 50° C. to about 95° C. The preferred time of the treatment step is from about 1 minute to about 180 minutes, more preferably from about 10 minutes to about 60 minutes. The preferred biomass for the method comprises, consists essentially of, or consists of soybean hull. It is preferred, but not required, that the hydrogel has a pectin-to-protein ratio of from about 0.1 to about 3.0. It is also preferred, but not required that cellulosic biomass is treated with the aqueous acidic solution at a liquid-to-solid ratio by mass of from about 1-to-1 to about 50-to-1, more preferably from about 5 to about 20.
Also disclosed is a hydrogel made by the method described and claimed herein.
SBH is one of the major byproducts of the soy industry, with a global annual production of approximately 20 million tons. More than 50% of that total is generated in the United States. This byproduct has much potential for high-value commercial utilization. The valorization of this low-cost biomass on an industrial scale could supplant its current low-value applications (mainly limited to animal feed). The unique chemical composition of SBH (high pectin and protein content) makes this biomass a promising candidate for hydrogel production. Most natural hydrogels are produced from pure/fractionated ingredients, such as pectin, protein, alginate, chitosan, and starch. The goal, achieved here, was to produce hydrogels from SBH without extracting specific components. The method thus results in more cost-effective and environmentally friendly processing with minimal effluent.
The properties of produced hydrogels are significantly affected by the applied processing conditions. Time, temperature, and pH of treatments were chosen as the independent factors, and a three-level face-centered central composite design (CCD) was used as the statistical method for the design of experiments (DOE). Selected levels of processing conditions are as follows: (T: 30-150° C., t: 1-180 min, pH: 1.0-6.0). The total number of trials was 20, including six replicates of the centroid. The effect of each processing condition and their interaction on the yield, water content, pectin/protein ratio, and viscoelasticity of the resulting hydrogels were evaluated using response surface methodology (RSM).
CCD=central composite design. GalA=D-galacturonic acid monohydrate. H=hydrogel with the highest pectin/protein ratio. Ip=isoelectric pH. L=hydrogel with the lowest pectin/protein ratio. LAOS=large amplitude oscillatory shear. LMP=low-methoxyl pectin. LVE=linear viscoelastic region. MC=moisture content. MHDP=3-phenylphenol (i.e., meta-hydroxy diphenyl). Pec/Pro=pectin/protein ratio. PAM=polyacrylamide. PCL=polycaprolactone. PEG=polyethylene glycol. pHEMA=poly-2-hydroxyethyl methacrylate. PLA=polylactic acid. PVA=polyvinyl alcohol. PVP=polyvinyl pyrrolidone. RSM=response surface methodology. SBH=soybean hull. SC=solids content. SPI=soy protein isolate. WHC=water-holding capacity.
All references to singular characteristics or limitations of the disclosed method shall include the corresponding plural characteristic or limitation, and vice-versa, unless otherwise specified or clearly implied to the contrary by the context in which the reference is made. The indefinite articles “a” and “an” mean “one or more.”
The word “about” when applied to a variable means+/−10% of the stated value.
The phrase “acidic solution” means a solution having a pH less than 7. The solution can be made acidic using any suitable acid, such as, but not limited to, acetic acid (CH3C(═O)OH), boric acid (B(OH)3), chloric acid (HCIO3), citric acid (CH2COOH—C(OH)COOH—CH2COOH), hydrobromic acid (HBr), hydrochloric acid (HCl), hydrofluoric acid (HF), hydroiodic acid (HI), nitric acid (HNO3), oxalic acid (HO(O═)CC(═O)OH), perchloric acid (HCIO4), sulfuric acid (H2SO4), and the like. HCl is preferred (but not required).
The word “biomass” is defined broadly herein to encompass all organic materials produced by plants and animals, such as cobs, husks, leaves, roots, seeds, shells, and stalks, as well as microbial and animal metabolic wastes (e.g., manure), without limitation. Common sources of biomass include (without limitation): (1) agricultural wastes, such as corn cobs and stalks, straw, seed hulls (including soybean hulls), sugarcane leavings, bagasse, nutshells, citrus peels, fruit and vegetable skins, egg shells, and manure from cattle, poultry, and hogs; (2) woody materials, such as wood or bark, sawdust, timber slash, and mill scrap; (3) municipal waste, such as waste paper and yard clippings; (4) energy crops, such as poplars, willows, switch grass, alfalfa, prairie bluestem, corn, soybean; and (5) coal, peat moss, and the like. The term “biomass-derived” refers to any reactant or material that can be fabricated from biomass by any means now known or developed in the future, including (without limitation) polysaccharides, monosaccharides, polyols, oxygenated hydrocarbons, sugars, starches, and the like.
The term “treating” refers to the act of touching, making contact, or of bringing to immediate or close proximity, including at the molecular level, for example, to bring about a chemical reaction, or a physical change, e.g., in a solution, in a mixture, or in a reaction mixture.
All combinations of method steps disclosed herein can be performed in any order, unless otherwise specified or clearly implied to the contrary by the context in which the referenced combination is made.
The method disclosed herein can comprise, consist of, or consist essentially of the essential elements and steps described herein, as well as any additional or optional ingredients, components, or limitations described herein or otherwise useful in organic chemistry.
Newly developed and disclosed herein is a method of producing a hydrogel from whole SBH. SBH is a pectin- and protein-rich biomass and can be used as a feedstock to make a hydrogel in single-step combining heat and acid gelation. Acid-extracted SBH pectin is a low-methyl pecting (LMP; i.e., a degree of methylation of <50%, according to recent literature data). See L. H. Reichembach, C. L. de Oliveira Petkowicz, New findings on acid-extractable pectins from soy hull, Carbohydr. Polym. 294 (2022) 119831. LMP gelation follows one of the two mechanisms: (I) electrostatic complex formation with divalent cations, mainly Ca2+, according to the egg-box model, and (II) conformational transition from an extended two-fold structure to a more compact three-fold structure. The egg-box model is more likely to happen when the pH is above the pKa of pectin, i.e., 3.5, with pectin in a more dissociated and ionized form. The conformational transition mechanism occurs at extremely low pH values where pectin is uncharged and electrostatic repulsion between carboxylic acid groups is suppressed, making them act as hydrogen donors, P. M. Gilsenan, R. K. Richardson, E. R. Morris, Thermally reversible acid-induced gelation of low-methoxy pectin, Carbohydr. Polym. 41 (2000) 339-349. Heat denaturation is usually a prerequisite for the gelation of globular protein (e.g., soy protein) because it unfolds the protein, cleaves the disulfide bonds, and exposes previously occluded polar side chains, as well as sulfhydryl and hydrophobic groups to the surface. These functional groups contribute to aggregate formation through covalent links (disulfide bonds) and non-covalent bonding (hydrogen bonding, electrostatic interactions, and hydrophobic interactions), followed by percolation to a 3D gel network. The interpolymer interactions and gelation mechanism/conditions depend on single biopolymers' inherited physicochemical and structural properties, the concentration ratio of constituting biopolymers, and extrinsic factors such as temperature, pH, shearing, and ionic strength. Here, the effect of pH, time, and temperature on the hydrogel yield, solids content, and pectin/protein (pec/pro) ratio was evaluated using central composite design (CCD) and response surface methodology (RSM), followed by the rheological analysis of the selected hydrogels.
Materials and Chemicals:
The materials and chemicals used were as follows: course ground SBH (Republic Mills, Inc., Napoleon, Ohio); sodium tetraborate (anhydrous, 99.5%) from BeanTown Chemical Corporation (Hudson, New Hampshire); D-galacturonic acid monohydrate (GalA, 97%) from Alfa Aesar (Ward Hill, Massachusetts); 3-phenylphenol (i.e., meta-hydroxy diphenyl “MHDP”) from TCI America (Portland, Oregon); sodium hydroxide (NaOH) pellets from Macron Fine Chemicals (Phillipsburg, New Jersey); hydrochloric acid (HCl, 36.5-38% Assay) and EDTA disodium salt dihydrate from BDH Chemicals (VWR, Radnor, Pennsylvania); sulfuric acid (H2SO4, 95-98% Assay) from J. T. Baker (Phillipsburg, New Jersey).
Experimental Design Using RSM:
To study how changing processing conditions (pH, time, and temperature) and their interactions affect the properties of the fabricated SBH-based hydrogel with a lower number of experimental runs and draw objective conclusions, experiments were designed using RSM. For this purpose, Minitab® software (Minitab, LLC., State College, Pennsylvania) was used by applying the face-centered CCD design option. Three factors of pH, temperature, and time were selected as the independent factors with three levels, with α=1. The total number of trials using RSM is 20 (with six repeats of the center point to ensure the model's repeatability) as opposed to the 27 trials required for the full factorial design. The details of the experimental runs are presented in Table 1, with low, high, and mid values of factors coded with −1, 1, and 0 (respectively) in the parentheses.
Reaction times, temperatures, and pH's extending beyond the values stated in Table 1 are explicitly with the scope of the method disclosed and claimed herein.
Hydrogel Fabrication:
A combination of thermochemical heat-induced and acid-induced gelation was used to fabricate hydrogels from SBH. First, an aqueous acidic dispersion of SBH was prepared by mixing the biomass with distilled water and concentrated HCl (for pH adjustment) at a liquid to solid (L/S) ratio of 9. Then the mixture was heated for 60 minutes, and immediately cooled down to room temperature in an ice bath. After this step, the heat- and acid-treated SBH was blended for 30 minutes (in 5 minutes intervals to ensure room-temperature blending). The resultant SBH mixture was kept at 4° C. overnight and centrifuged at 9500 RPM for 15 minutes to separate it into three fractions: solid, hydrogel, and liquid. Although, the hydrogel fraction is the focus, it is worth noting that besides the main hydrogel product, the separated insoluble fibrous solids and solubilized liquid fractions can be used as precursors of cellulose/cellulose derivatives and films/coating production, respectively, making the integrated multi-product approach highly economical and scalable.
Yield, Solids Content, and Composition of Hydrogel:
Each of the twenty hydrogels produced according to Table 1 were tested for their yield, solids content and pectin and protein percentage. The yield (%) was calculated based on the wet weight of hydrogels multiplied by the corresponding solids content divided by the initial SBH weight. The solids content (%) of the samples was calculated using the following formula: SC (%)=100-MC (%), where SC and MC are solids and moisture contents, respectively. MC of hydrogels was determined by drying 5 to 6 grams of each sample in a HC 103 moisture analyzer (Metier Toledo, Columbus, Ohio) operating at 111° C.
Quantifying the pectin and protein content of hydrogels was done on the freeze-dried samples. Pectin content was determined as GalA content according to the modified “MHDP” (meta-hydroxy diphenyl, i.e., 3-phenylphenol) colorimetric method. P. K. Kintner Ill, J. P. Van Buren, Carbohydrate interference and its correction in pectin analysis using the m-hydroxydiphenyl method, J. Food Sci. 47 (1982) 756-759. The absorbance of samples was measured using a Thermo Scientific™ GENESYS™ 10S UV-Vis spectrophotometer, and D-GalA standards of 0-100 mg/L were used for calibration curve generation. Protein content was determined indirectly through nitrogen content (N) analysis by the combustion method using a TruMac Nitrogen Analyzer (Leco Corporation, St. Joseph, Michigan) and protein conversion factor (protein content=N*6.25) with EDTA as the standard. The pec/pro ratio was calculated for each sample based on the values obtained through the experimental techniques noted herein.
Response Surface Regression:
A full quadratic model including all individual factors (pH, time, temperature), squared terms (pH*pH, time*time, temperature*temperature), and 2-way interaction terms (pH*time, pH*temperature, time*temperature) was used to conduct a regression analysis on yield and pec/pro ratio as the selected responses. The regression models, corresponding residual plots, and statistically significant factors were obtained from the Minitab software model reports.
Rheological Characterization:
The rheological behavior of selected SBH hydrogels was tested using a stress-controlled magnetic-bearing rheometer (AR-G2, TA Instruments, New Castle, Delaware) equipped with a 40-mm parallel plate at a gap size of 500 μm. (See J. Yang, M. Shen, T. Wu, Y. Luo, M. Li, H. Wen, J. Xie, Role of salt ions and molecular weights on the formation of Mesona chinensis polysaccharide-chitosan polyelectrolyte complex hydrogel, Food Chem. 333 (2020) 127493.) All tests were done using a solvent trap to avoid moisture loss during analysis. A strain sweep test was performed at 25° C. from 0.1% to 100% at a constant angular frequency of 1 rad/s to determine the linear viscoelastic region (LVE) for samples. Based on the results, the frequency sweep test was performed at a constant strain of 0.3%, 25° C. and 0.1 to 100 rad/s. The steady-state test was conducted to evaluate the flow behavior of hydrogel samples under shear rates of 0.1-100 (1/s) at a temperature of 25° C.
Hydrogel Yield and Composition:
The hydrogel production yield, solids content, and the percent weight of pectin and protein in each gel was determined. The results are presented in Table 2. The sample numbers and the corresponding processing conditions are the same as the run orders in the experimental design (Table 1). The results show the significant effect of processing conditions on the hydrogel formation efficiency, with yields ranging from 1.54% to 29.37% and solids contents of 8-16.55%. Pectin (GalA) and protein contents in the fabricated hydrogels ranged from 10.00% to 19.58% and 18.29 to 22.36%, respectively, resulting in pec/pro ratios of from 0.47 to 1.03. To better understand the effect of processing conditions on the hydrogel properties, yield (the combined contribution of weight and solids content) and pec/pro ratio (the combined contribution of pectin and protein content) were selected as model responses for regression using RSM.
Response Surface Regression:
Yield:
Regression analysis of yield versus pH, time, and temperature was conducted based on the experimental values obtained for the 20 hydrogels using the Minitab software, and the following regression equation (1) was generated by the software:
Yield(%)=−28.9−7.15 pH+0.708 Time+0.85 Temperature+1.086 pH*pH−0.00950 Time*Time−0.0024 Temperature*Temperature−0.0072 pH*Time−0.0413 pH*Temperature+0.00026 Time*Temperature Equation (1)
The equation represents all terms for a full quadratic model, i.e., linear, square, and 2-way interactions. The reported coefficient of determination (R2) is 85.17%. However, as depicted in the normal plot of the standardized effect,
Yield(%)=−12.39−2.453pH+0.546Time+0.3203Temperature−0.00728Time*Time Equation (2)
The significant model factors affecting the yield (%) are labeled in
Increased acidity and higher temperatures are reported to increase pectin extraction from the middle lamella in the cell wall. F. Gutohrlein, S. Drusch, S. Schalow, Extraction of low methoxylated pectin from pea hulls via RSM, Food Hydrocoll. 102 (2020) 105609. As for protein, alkaline or acidic conditions far away from the isoelectric pH (Ip) can result in better protein extraction from biomass due to the increased net charge and improved solubility. H. Kamal, C. F. Le, A. M. Salter, A. Ali, Extraction of protein from food waste: An overview of current status and opportunities, Compr. Rev. Food Sci. Food Saf. 20 (2021) 2455-2475. Soy protein has two major subunits, glycinin (11S) and β-conglycinin (βCG, 7S), with neutral pH denaturation temperatures of 90° C. and 74° C., respectively. The denaturation temperatures shift to lower values with a decrease in pH level. Therefore, higher temperature combined with higher acidity ensures the gelation of both subunits and increases the soy protein contribution to the final hydrogel. Also, higher temperatures increase the aggregate formation and growth rate, resulting in faster gel formation, as reported by Nicolai and Chassenieux (2019) on investigating the heat-induced globular protein gelation. T. Nicolai, C. Chassenieux, Heat-induced gelation of plant globulins, Curr. Opin. Food Sci. 27 (2019) 18-22. In terms of the effect of interpolymer interactions, low pH and increased temperature favor pectin de-esterification and an increase the number of free carboxylic acid groups on pectin's surface. This can improve the hydrogen bonding to amide groups in soy protein at low pH and increase the overall hydrogel yield. This is consistent with earlier observations on acid-induced gelation of amidated LMP. See M. Dominiak, K. M. Sdndergaard, J. Wichmann, S. Vidal-Melgosa, W. G. T. Willats, A. S. Meyer, J. D. Mikkelsen, Application of enzymes for efficient extraction, modification, and development of functional properties of lime pectin, Food Hydrocoll. 40 (2014) 273-282 and F. Capel, T. Nicolai, D. Durand, P. Boulenguer, V. Langendorff, Calcium and acid induced gelation of (amidated) low methoxyl pectin, Food Hydrocoll. 20 (2006) 901-907. Also, the higher degree of protein denaturation afforded under acidic conditions and elevated temperatures provides a higher probability of hydrophobic interactions between the exposed nonpolar groups of protein and available acetyl/methyl groups of pectin. However, due to the limited availability of the hydrophobic groups in the case of SBH pectin, hydrophobic interactions are more likely of a protein-protein type, with less contribution attributed to the pectin-protein association.
The hydrogel yield is inversely correlated to the heating time, and the dependency is of the second order (BB term in
The normal probability plot (
Among these factors, ionic strength is the only extrinsic factor, and its addition to the model could enhance the predictability of hydrogel formation yield. SBH has large amounts of minerals in its structure. These minerals could be released into the soluble fraction during the combined heat and acid treatments. See Table 9. Some of the possible influences of these cations on the gelation mechanism could be the adverse effect on electrostatic interpolymer bonding when pKa of pectin<pH<Ip of protein (i.e., pH=3.8) by electrostatic shielding of the negative charges on pectin, neutralization of negative charge on proteins at pH>Ip, decrease or increase in the contribution of LMP gelation through electrostatic bonding with divalent cations, especially Ca2+, and forming egg-box model in pH>pKa of pectin (i.e., pH=3.8 and 5.8), as well as inhibiting protein-protein or pectin-pectin electrostatic repulsion when they carry the same charge.
Approximately 0.50-10 g dried solid samples were weighed and transferred into ceramic crucibles. The crucibles were placed in a muffle furnace with a set temperature of 500° C. Samples were kept in the furnace overnight, allowed to cool down, and digested by mixed solution of 1 N nitric acid and 1 N hydrochloric acid on a hot plate at ˜168° C. Hydrolysate were separated using a Whatman No. 1 filter paper and brought to volume in a 100 ml volumetric flask using deionized water. Finally, the resultant solutions were analyzed by Inductively Coupled Plasma Optical Emission Spectroscopy (Vista-MPX ICP-OES Radial Spectrometer, Varian Inc., USA) calibrated with standard concentrations of the desired elements. (Odom, John W., and Mama B. Koné. “Elemental analysis procedures used by the Auburn University Department of Agronomy and Soils.” (1997).)
Another determinant factor in decreasing the predictability of hydrogel yield is that the calculation is based on the solids content and hence, the water holding capacity (WHC) of the hydrogel matrix. Because factors like the interpolymer and polymer-water interactions, conformational arrangements of pectin/protein, and their morphologies can significantly influence the WHC of the fabricated hydrogel, the predictability of its behavior solely based on the processing conditions can add uncertainty and lack of fit to the yield as a model response.
Using the pec/pro ratio calculated based on the pectin and protein contents (%) of hydrogels (Table 2) as the model response, a full quadratic regression was performed to evaluate the effect of processing conditions. The following equation (3) was reported by the Minitab software:
Pec/Pro Ratio=−0.791−0.2779pH+0.00667Time+0.0402Temperature+0.02401pH*pH+0.000084Time*Time−0.000183Temperature*Temperature+0.000193pH*Time+0.000894pH*Temperature−0.000210Time*Temperature Equation 3
Although the pec/pro model shows more statistically significant terms in the model than the yield regression (
The decrease in R2 before and after implying the backward elimination was smaller than that of the yield regression analysis, showing higher goodness of fit and predictability for the pec/pro ratio. This is also reflected in much lower residual values of the model for this response in
Pec/Pro Ratio=0.042−0.2348pH+0.01330Time+0.01429Temperature+0.02864pH*pH−0.000210Time*Temperature Equation 4
The higher accuracy of pec/pro response is mainly because it is calculated based on the contents of these biopolymers, as determined by quantifying the GalA and nitrogen content. The calculation thus excludes (a) the role of monosaccharide composition; (b) the distribution of GalA in the backbone; (c) the acetyl/methyl contents for pectin; (d) the amino acid composition; (e) the amount of acidic and basic subunits; and (f) the extent of denaturation and surface hydrophobicity for protein. Also, this response is much less dependent on the gel properties, such as WHC and interpolymer interactions, compared to the yield. (Adding ionic strength as an additional independent factor to the model might further improve the predictability of the pec/pro ratio.)
Due to the better goodness of fit and predictability of the pec/pro ratio compared to the yield, this response was the basis of sample selection for evaluating the rheological behavior of the hydrogels. Rheological analysis of the hydrogels with the highest (H) and lowest (L) pec/pro ratio was undertaken to gain insight into the gel strength and flow behavior of the fabricated novel hydrogels.
Rheological Analysis:
The strength and flow properties of hydrogels H (highest pec/pro ratio; 1.03; entry 9 of Table 2) and L (lowest pec/pro ratio; 0.47— entry 17 of Table 2) were tested according to procedures described hereinabove in the section titled “Rheological Characterization.” The results are presented in
The flow behavior of the hydrogels was evaluated by applying steady shear of 0.1-100 1/s (
As disclosed herein, novel pectin-protein hydrogels were produced from SBH through hydrothermal acidic gelation of pectin and protein without requiring individual biopolymer isolation and purification. The separated insoluble solid and liquid phases are rich in cellulose and dissolved non-gelling fraction of polymers, which can be used to produce cellulose specialties and films/coatings, respectively. The effect of pH, time, and temperature on gel yield and pec/pro ratio was studied using CCD and RSM. The regression analysis showed that all factors were statistically significant in both responses. No significant contribution of the interaction of these factors was observed on the yield, while time*temperature was the only crucial 2-way interaction term affecting the pec/pro ratio.
Higher temperatures and lower pH positively affected the extraction and gelation of pectin and protein. While increasing the heating time facilitated depolymerization and subsequent reduction in the yield. Increasing temperature and pH positively influenced the pec/pro ratio with increased pectin extraction and less degradation of the GalA in the backbone. The lower pH (far from the protein's Ip) is a prerequisite for protein solubility and increasing its contribution to the gel phase. In comparison, protein would form heat-induced insoluble aggregates at higher pH (close to the Ip), which will mostly stay with the insoluble solid fraction after centrifugation.
The results of regression modeling revealed that the pec/pro ratio was more predictable with higher goodness of fit (based on the R2 and residual values) because it is mainly affected by the studied factors rather than being dependent on the intrinsic structure, conformational, and morphological polymer properties and the dynamic interpolymer interactions, which is the case for hydrogel yield. Since the RSM more accurately predicted the pec/pro ratio, the hydrogels with the highest and lowest pec/pro ratio (H and L) were selected to evaluate hydrogel properties through rheological tests.
Both hydrogels can be classified as physical gels with relatively low mechanical strength (yield strains of approximately 1.6% and 1.0% for L and H), frequency-dependent storage and loss moduli, and shear thinning behavior. Hydrogel L had a higher protein concentration, mostly in the clustered large aggregates form resulting in a stiff opaque particulate gel with inferior WHC. The pectin-protein interaction was mostly of ionic type between the oppositely charged biopolymers at pH=3.8. Therefore, hydrogel L showed higher strength, viscosity, and yield strain. However, the thermodynamic incompatibility between pectin and protein introduced a high level of heterogeneity and brittleness to the gel structure, reflected in two-step yielding and cascade changes in its moduli as well as the appearance of lower and higher shear thinning regions at low and high shear rates. Hydrogel H, conversely, had a creamy texture, with fine-stranded homogenous structure and higher WHC due to the smaller particle sizes of both biopolymers and the more available interparticle void space for water entrapment. Therefore, its shear- and strain-induced flow behaviors were more monotonous. However, due to biopolymers' lower molecular weight and non-ionic interactions, hydrogel H had lower viscosity and strength.
The results show tunable functional properties can be achieved for the novel hydrogels with applications in the food and cosmetics industry due to the similarity of the rheological behavior to the food and emulsion gels.
Priority is hereby claimed to provisional application Ser. No. 63/425,074, filed Nov. 14, 2022, which is incorporated herein by reference.
This invention was made with government support under 58-6010-0-010 awarded by the U.S. Department of Agriculture. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63425074 | Nov 2022 | US |
