Effects of water-soluble soybean polysaccharide on rheological properties,stability and lipid digestibility of oil-in-water emulsion during in vitro gastrointestinal digestion

Water-soluble soybean polysaccharide (SSPS) is a naturally occurring emulsifier. SSPS was used as the sole emulsifier to stabilize an oil-in-water (O/W) emulsion. The effects were investigated of different SSPS concentrations (3–20% (w/w)) on the lipid digestibility, rheological properties and stability of O/W emulsions during in vitro digestion model. The droplet size of the emulsions tended to increase during the oral phase because the emulsions were unstable and droplets coalesced, except with a SSPS concentration of 20% (w/w). The presence of SSPS markedly reduced the free fatty acid (FFA) content after its stabilized O/W emulsion passed through in vitro gastrointestinal digestion. The amount of FFA significantly decreased as the concentration of SSPS increased due to SSPS stabilization film on oil droplet surface and high viscous system. SSPS may be an attractive alternative ingredient to control the lipid digestibility of emulsions for various food products.     

赋形剂乳液粒径和油脂链长对橘子中β-胡萝卜素的物化特性及生物可给性的影响

利用体外模拟胃肠道(Gastrointestinal tract,GIT)消化模型,研究了粒径大小和油脂链长对赋形剂乳液/橘子混合体系在胃肠道消化过程中物化特性、微观形态的变化和对橘子中β-胡萝卜素生物可给性的影响。结果表明:相比于中等粒径(500 nm)乳液和大粒径(10 μm)乳液,小粒径(200 nm)乳液的物理特性(粒径和电位)和微观形态在各个模拟消化阶段中的变化趋势最明显;除小肠消化阶段之外,中链油脂(以MCT油为代表)制备的赋形剂乳液的物理特性与长链油脂(以玉米油为代表)制备的赋形剂乳液没有显著性差异(p>0.05);小粒径乳液的油脂消化速率最快,其生物可给性提升(38.13%)的效果显著大于中粒径(24.93%)和大粒径(26.23%)乳液(p<0.05);长链油脂与中链油脂的油脂消化速率的差异不显著(p>0.05);与中链油脂相比,长链油脂制备的赋形剂乳液对提高橘子中β-胡萝卜素的生物可给性具有更显著的影响(p<0.05)。研究结果对于科学设计赋形剂乳液来提高果蔬中亲脂性生物活性物质的生物可给性具有重要指导作用。     

Characterization of Chemically and Thermally Treated Oil‐in‐Water Heteroaggregates and Comparison to Conventional Emulsions

Heteroaggregated oil‐in‐water (O/W) emulsions formed by targeted combination of oppositely charged emulsion droplets were proposed to be used for the modulation of physical properties of food systems, ideally achieving the formation of a particulate 3‐dimensional network at comparably low‐fat content. In this study, rheological properties of Quillaja saponins (QS), sugar beet pectin (SBP), and whey protein isolate (WPI) stabilized conventional and heteroaggregated O/W emulsions at oil contents of 10% to 60% (w/w) were investigated. Selected systems having an oil content of 30% (w/w) and different particle sizes (d₄₃ ≤ 1.1 or ≥16.7 μm) were additionally subjected to chemical (genipin or glutaraldehyde) and thermal treatments, aiming to increase network stability. Subsequently, their rheological properties and stability were assessed. Yield stresses (τ₀) of both conventional and heteroaggregated O/W emulsions were found to depend on emulsifier type, oil content, and initial droplet size. For conventional emulsions, high yield stresses were only observed for SBP‐based emulsions (τ₀,_SBP approximately 157 Pa). Highest yield stresses of heteroaggregates were observed when using small droplets stabilized by SBP/WPI (approximately 15.4 Pa), being higher than those of QS/WPI (approximately 1.6 Pa). Subsequent treatments led to significant alterations in rheological properties for SBP/WPI systems, with yield stresses increasing 29‐fold (glutaraldehyde) and 2‐fold (thermal treatment) compared to untreated heteroaggregates, thereby surpassing yield stresses of similarly treated conventional SBP emulsions. Genipin‐driven treatments proved to be ineffective. Results should be of interest to food manufacturers wishing to design viscoelastic food emulsion based systems at lower oil droplet contents.     

A Novel Approach to Improving the Quality of Bittern-Solidified Tofu by W/O Controlled-Release Coagulant. 1: Preparation of W/O Bittern Coagulant and Its Controlled-Release Property

To explore the controlled-release capacity and potential application of water in oil (W/O) emulsions, W/O emulsions with bittern solution as the aqueous phase were prepared with polyglycerol polyricinoleate (PGPR) as the emulsifier. Several factors that influenced the preparation of W/O bittern coagulants were investigated. Optimized conditions were applied as follows: bittern solution/oil phase at 4:6 or 5:5 (w/w), processing temperature at 65 °C, emulsifier concentration higher than 0.6 wt% and high-pressure homogenization at 60 MPa; W/O emulsions that had a good particle size distribution, good rheological properties, good embedding ratio and high stability were prepared. The optimized W/O emulsions also exhibited the expected controlled-release ability, affected by the bath temperature, shear speed and emulsion characteristics. The preliminary results also showed that the application of W/O emulsions has the potential to improve tofu quality by delaying the solidification of soybean protein and creating a much smoother and finer gel structure.     

In vitro bioaccessibility of ergocalciferol in nanoemulsion‐based delivery system: the influence of food‐grade emulsifiers with different stabilising mechanisms

The effect of emulsifier type on the in vitro bioaccessibility of ergocalciferol‐loaded nanoemulsions was examined (mouth, stomach and small intestinal phases). Oil‐in‐water nanoemulsions were prepared using emulsifiers with different stabilising mechanisms: decaglycerol monooleate (MO7S; steric), modified lecithin (ML; electrostatic), sodium caseinate (SC; electrosteric) and ML‐MO7S (combined electrostatic and steric). The droplet size, size distribution, ζ‐potential and microstructure of nanoemulsions during digestion depended on the emulsifier type. The fate of lipid in the small intestinal phase also relied on the emulsifier type, with the free fatty acids release rate decreasing in the following order: MO7S > ML‐MO7S > ML > SC. The ergocalciferol bioaccessibilities in nanoemulsions prepared using MO7S (62%), ML (64%) and ML‐MO7S (65%) were similar and significantly higher than that stabilised by SC (12%). No significant loss of ergocalciferol was observed in all nanoemulsions after full digestion; they were chemically stable against digestion conditions, regardless of the emulsifier type.     

Stability and release properties of double-emulsions stabilised by caseinate–dextran conjugates

The subject of the present paper was to investigate the possibility of stabilising water-in-oil-in-water emulsions (W/O/W) by using sodium caseinate (SC)–dextran (Dex) conjugates in order to influence the release of vitamin B₁₂ from the inner water phase (W₁) to the outer aqueous phase (W₂).To prepare the conjugate the SC was combined with Dex (M_r 250,000 or 500,000 g/mol) and incubated at 60 °C and a humidity of 79% for 8 h.The double emulsions, with encapsulated vitamin B₁₂, were prepared using a two-step emulsification technique. Whereas different amounts of polyglycerin polyricinoleate (PGPR, E476) were the hydrophobic emulsifier, the conjugate and the SC alone were used as the hydrophilic emulsifiers. The investigations comprised the determination of the particle size distribution of the W/O/W emulsion and measurement of the amount of vitamin B₁₂ migration from W₁- to the W₂-phase during the second stage of emulsion preparation and after heating or pH changing of emulsion.The water-containing oil droplets of the W/O/W emulsions were smaller and distributed more narrowly using SC–Dex conjugate as emulsifier instead of pure protein. Under acidic conditions, the conjugate-containing emulsions were more coalescence stable than the emulsions with SC, and the vitamin B₁₂ release from the inner W₁-phase was significantly decreased.     

Synergistic effects of polyglycerol ester of polyricinoleic acid and sodium caseinate on the stabilisation of water–oil–water emulsions

The inherent thermodynamic instability of water–oil–water (W/O/W) emulsions has restrictions for their application in food systems. The objective of this study was to develop a food grade W/O/W emulsions with high yield and stability using minimal concentrations of surfactants. Emulsions were prepared using soybean oil, polyglycerol ester of polyricinoleic acid (PGPR) alone or in combination with sodium caseinate (NaCN) as emulsifier(s) for primary water-in-oil (W/O) emulsions and NaCN as the sole emulsifier for secondary W/O/W emulsions. Increasing the concentration of PGPR (0.5–8%w/v) had no effect on the droplet sizes of the resulting W/O/W emulsions. However, significant increases in droplet sizes of W/O/W emulsions were observed when the concentration of NaCN in external phase was reduced from 0.5 to 0.03% (w/v) (p<0.05). Percentage yields of emulsions (using a water-soluble dye) improved when PGPR concentration in the inner phase was increased from 0.5 to 8% (w/v). A stable W/O/W emulsion with a yield >90% could be prepared with 4% (w/v) PGPR alone as primary hydrophobic emulsifier and 0.5% (w/v) NaCN as external hydrophilic emulsifier. The concentration of PGPR in the inner phase could be reduced to 2% (w/v) without affecting the yield and stability of the W/O/W emulsion by partially replacing PGPR with 0.5% (w/v) NaCN, which was added to the aqueous phase of the primary W/O emulsion. The results indicate that a possible synergistic effect may exist between PGPR and NaCN, thus allowing formulation of double emulsions with reduced surfactant concentration.     

Influence of lipid type on gastrointestinal fate of oil-in-water emulsions: In vitro digestion study

The potential gastrointestinal fate of oil-in-water emulsions containing lipid phases from different sources was examined: vegetable oils (corn, olive, sunflower, and canola oil); marine oils (fish and krill oil); flavor oils (orange and lemon oil); and, medium chain triglycerides (MCT). The lowest rates and extents of lipid digestion were observed for emulsified flavor oil, followed by emulsified krill oil. There was no appreciable difference between the final amounts of free fatty acids released for emulsified digestible oils. Differences in the digestibility of emulsions prepared using different oils were attributed to differences in their compositions, e.g., fatty acid chain length and unsaturation. The particle size distribution, particle charge, microstructure, and macroscopic appearance of the emulsions during passage through the simulated GIT depended on oil type. The results of this study may facilitate the design of functional foods that control the digestion and absorption of triglycerides, as well as the bioaccessibility of hydrophobic bioactives.     

Relation between in vitro lipid digestion and β-carotene bioaccessibility in β-carotene-enriched emulsions with different concentrations of l-α-phosphatidylcholine

In view of consumer health, it is desirable to promote the bioaccessibility of lipid-soluble compounds like carotenoids, while limiting the lipid intake. The objective of this work was to examine the relation between in vitro lipid digestion and β-carotene bioaccessibility of carrot-based model food emulsions containing water, 5% olive oil enriched with β-carotene (from carrots) and different concentrations (1–2–3–4%) of l-α-phosphatidylcholine (PHC), as an emulsifier. The lipid digestion (hydrolysis of triacylglycerols (TAGS) and incorporation of free fatty acids (FFAs) and monoacylglycerols (MAGs) in the micelles) and the β-carotene bioaccessibility (incorporation of β-carotene in the micelles) were studied after an in vitro digestion procedure wherein the stomach phase was mimicked for 2.0 h (37 °C) and the small intestinal phase was mimicked for 1.0 h, 1.5 h and 2.0 h (37 °C) (both end-over-end rotations). As a consequence, not only the influence of the emulsifier concentration, but also the influence of the duration in the small intestinal phase was investigated in this study. The oil droplet size distributions of the emulsions at different stages of digestion were shown to be dependent on the phosphatidylcholine concentration, but independent on the duration in the small intestinal phase (1.0 h–2.0 h). Furthermore, all TAGs were already hydrolysed into FFAs and MAGs after 1.0 h small intestinal phase and the incorporation of FFAs and MAGs into micelles seemed to reach a maximum for all emulsions (approximately 26.5%), independent on the phoshpatidylcholine concentration and thus on the particle size distributions. Finally, the β-carotene bioaccessibility increased with increasing phosphatidylcholine concentration, ranging from 33.2% to 79.8% for a 1% and 4% PHC emulsion respectively. No significant differences in β-carotene bioaccessibility were however noticed for the different durations in the small intestinal phase tested. In conclusion, a higher phosphatidylcholine concentration in emulsions leads to higher β-carotene bioaccessibility while the incorporation of lipids into micelles did not increase.     

Formulation of monodisperse oil‐in‐water emulsions loaded with ergocalciferol and cholecalciferol by microchannel emulsification: insights of production characteristics and stability

Nutritional deficiencies of ergocalciferol (VD₂) and cholecalciferol (VD₃) cause skeletal deformations. The primary aim of this study was to encapsulate VD₂ and VD₃ in food‐grade oil‐in‐water (O/W) emulsions by using microchannel emulsification (MCE). Silicon asymmetric straight‐through microchannel (MC) array consisting of 10 313 channels, each having an 11 × 104 μm microslot connected to a 10 μm circular microholes. 1% (w/w) sodium cholate or Tween 20 in water was used as the continuous phase, while 0.5% (w/w) of each VD₂ and VD₃ in different oils served as the dispersed phase. Monodisperse O/W emulsions with Sauter mean diameters of 28 to 32 μm and relative span factor widths below 0.3 were formulated via an asymmetric straight‐through MC array under appropriate operating conditions. The monodisperse O/W emulsions stabilised with Tween 20 remained stable for >30 days with encapsulation efficiencies (EEs) of VD₂ and VD₃ of above 70% at 4 and 25 °C. In contrast, those stabilised with sodium cholate had stability of >30 days with their EEs of over 70% only at 25 °C.     

The Effect of Sodium Carboxymethylcellulose on the Stability and Bioaccessibility of Anthocyanin Water-in-Oil-in-Water Emulsions

Water-in-oil-in-water (W₁/O/W₂) emulsions provide protective encapsulation to plant bioactive compounds in food matrix and under gastrointestinal conditions. However, the stability of the emulsions during the storage is crucial for their use in the food industry. Hence, the aim of this study was to enhance the stability and bioaccessibility of W₁/O/W₂ emulsions containing anthocyanins with the use of sodium carboxymethylcellulose (CMCNa). The emulsions were prepared by ultrasound technology, adding polyglycerol polyricinoleate (PGPR) in the inner aqueous phase of emulsions, and lecithin and Tween 20 in the outer aqueous phase. The systems were physicochemical characterized over the time and their behavior under simulated gastrointestinal conditions was investigated. Our results showed high encapsulation efficiencies above 90% and an increase in bioaccessibility with the use of CMCNa. Moreover, the polymer addition slowed down the free fatty acid release and increased the oil digestibility of lecithin-stabilized emulsions. These latter emulsions presented the highest bioaccessibility (31.08?±?1.73%), the more negative values of ζ-potential and no variations on the particle size and the backscattering profile over the time, thus being the most stable emulsions. These results provide useful information for the design of anthocyanin emulsion-based delivery systems to guarantee their functionality in food matrices as well as through the gastrointestinal tract.     

Effects of different nut oils on the structures and properties of gel-like emulsions induced by ultrasound using soy protein as an emulsifier

Gel-like emulsions were produced by high-intensity ultrasound (HIU) emulsification using soy protein as an emulsifier. Sunflower (a seed kernel oil), peanut (a groundnut oil), hazelnut, walnut, almond and pine nut oils were chosen as oil phases. The kinds of nut oil significantly affect the structures and properties of gel-like emulsions. The particle size of sunflower oil gel-like emulsion was smallest. Moreover, the macrorheological and microrheological results indicated that the values of apparent viscosity, storage modulus (G′) and elasticity index (EI) of sunflower and almond oil gel-like emulsions were higher while those of the walnut and pine nut oil gel-like emulsions were lower. Moreover, the sunflower oil gel-like emulsion was more stable after 2-month storage. The current study illustrated that HIU was of great importance for the production of high-viscosity gel-like emulsions. Furthermore, the differences of nut oils resulted in diverse physicochemical properties of gel-like emulsions.     

Preparation and impact of multiple (water-in-oil-in-water) emulsions in meat systems

The aim of this paper was to prepare and characterise multiple emulsions and assess their utility as pork backfat replacers in meat gel/emulsion model systems. In order to improve the fat content (in quantitative and qualitative terms) pork backfat was replaced by a water-in-oil-in-water emulsion (W₁/O/W₂) prepared with olive oil (as lipid phase), polyglycerol ester of polyricinoleic acid (PGPR) as a lipophilic emulsifier, and sodium caseinate (SC) and whey protein concentrate (WP) as hydrophilic emulsifiers. The emulsion properties (particle size and distribution, stability, microstructure) and meat model system characteristics (composition, texture, fat and water binding properties, and colour) of the W₁/O/W₂, as affected by reformulation, were evaluated. Multiple emulsions showed a well-defined monomodal distribution. Freshly prepared multiple emulsions showed good thermal stability (better using SC) with no creaming. The meat systems had good water and fat binding properties irrespective of formulation. The effect on texture by replacement of pork backfat by W₁/O/W₂ emulsions generally depends on the type of double emulsion (associated with the hydrophilic emulsifier used in its formulation) and the fat level in the meat system.     

Effects of emulsifier charges on the oxidative stability in oil-in-water emulsions under riboflavin photosensitization

The oxidative stability in oil-in-water (O/W) emulsions containing different emulsifier charges was tested under riboflavin photosensitization by analysis of headspace oxygen content and lipid hydroperoxides. Sodium dodecyl sulfate (SDS), Tween 20, and cetyltrimethylammonium bromide (CTAB) were selected as anionic, neutral, and cationic emulsifiers, respectively. The O/W emulsions containing CTAB had lower oxidative stability than those with SDS and Tween 20. The addition of ethylenediaminetetraacetic acid, a well-known metal chelator, increased the oxidative stability in O/W emulsions, irrespective of emulsifier charges. Oxidative stability in Tween 20-stabilized emulsions decreased in FeCl₃ and FeCl₂ concentration-dependent manner. However, oxidative stability in samples containing CTAB increased up to 0.5mM of FeCl₃ and FeCl₂ and then decreased, which implies that CTAB act differently during lipid oxidation compared to SDS and Tween 20.     

The characterization of the semi-solid W/O/W emulsions with low concentrations of the primary polymeric emulsifier

Semi-solid multiple W/O/W emulsions with low concentrations (0.8, 1.6 and 2.4% w/w) of lipophilic polymeric primary emulsifier PEG-30-dipolyhydroxystearate (PDHS) have been formulated. Both emulsions, primary and multiple, were prepared with high content of inner phase (Phi1 = Phi2 = 0.8). All the formulations differ only in the lipophilic emulsifier concentration. Evaluating several parameters such as macroscopic and microscopic aspect, droplet size, accelerated stability under centrifugation and flow and oscillatory rheological behaviour, assessed the multiple systems. It is possible to formulate the semi-solid W/O/W multiple emulsions with low concentrations of PDHS as the primary emulsifier. It appeared that the highest long-term stable multiple emulsion with the lowest droplet size, the highest apparent viscosity and highest elastic characteristic, was the sample with the highest concentration (2.4% w/w) of the primary emulsifier.     

重质碳酸钙稳定的O/W型Pickering乳液及其粒度效应研究

以重质碳酸钙作为颗粒稳定剂制备O/W型Pickering乳液,利用球磨法获得不同尺寸的碳酸钙,研究碳酸钙颗粒粒度、颗粒浓度、油相比例对乳液类型、稳定性、微观形貌及流变学特性的影响。结果表明:球磨后不同粒度的碳酸钙均能稳定Pickering乳液,且该乳液具有一定的储存稳定性,固体颗粒粒径对乳液性质有一定的影响,乳液粒径随固体颗粒粒径的增大而增大;随固体颗粒浓度的增大,乳液的乳析指数、乳液粒径以及乳液黏度均减小,但当固体颗粒浓度达到9g/100 mL以上时,增加颗粒浓度对乳液粒径影响不大;增大油相比例会使乳析指数减小、乳液粒径和黏度增大。     

Emulsifying properties of collagen fibers: Effect of pH,protein concentration and homogenization pressure

The emulsifying properties of collagen fibers were evaluated in oil-in-water (O/W) emulsions produced under different conditions of pH, protein content and type of emulsification device (rotor–stator and high-pressure homogenizer). The stability, microstructure and rheology of the O/W emulsions were measured. The phase separation and droplet size of the emulsions prepared using the rotor–stator device (primary emulsion) decreased with protein concentration and reduction in pH, allowing the production of electrostatically stable emulsions at pH 3.5. In contrast, emulsions at higher pH values (4.5, 5.5 and 7.5) showed a microscopic three-dimensional network responsible for their stability at protein contents higher than 1.0% (w/w). The emulsions at pH 3.5 homogenized by high pressure (up to 100 MPa) showed a decrease in surface mean diameter (d₃₂) with increasing pressure and the number of passes through the homogenizer. These emulsions showed droplets with lower dispersion and d₃₂ between 1.00 and 4.05 μm, six times lower than values observed for primary emulsions. The emulsions presented shear-thinning behavior and lower consistency index and viscosity at higher homogenization pressures. In addition, the emulsions showed a less structured gel-like behavior with increase in homogenization pressure and number of passes, since the pressure disrupted the collagen fiber structure and the oil droplets. The results of this work showed that the collagen fiber has a good potential for use as an emulsifier in the food industry, mainly in acid products.     

Release rate profiles of magnesium from multiple W/O/W emulsions

Water-in-oil-in-water (W/O/W) emulsions were formulated based on rapeseed oil, olive oil, olein and miglyol. Polyglycerol polyricinoleate and sodium caseinate were used as lipophilic and hydrophilic emulsifiers, respectively. Magnesium was encapsulated in the inner aqueous droplets. Emulsion stability was assayed through particle sizing and magnesium release at two storage temperatures (4 and 25 °C) over 1 month. Irrespective of the oil nature, both the primary W/O and W/O/W emulsions were quite stable regarding the size parameters, with 10-μm fat globules and 1-μm internal water droplets. Magnesium leakage from W/O/W emulsions was influenced by the oil type used in the formulation: the higher leakage values were obtained for the oils characterized by the lower viscosity and the higher proportion of saturated fatty acids. Magnesium release was not due to droplet–globule coalescence but rather to diffusion and/or permeation mechanisms with a characteristic rate that varied over time. In addition, W/O/W emulsions were resistant to various thermal treatments that mimicked that used in pasteurization processes. Finally, when W/O/W emulsions were placed in the presence of pancreatic lipase, the emulsion triglycerides were hydrolysed by the enzyme. These results indicated a possible use of W/O/W emulsions loaded with magnesium ions in food applications.     

超声法制备婴儿配方营养油乳液工艺的优化

为了降低婴儿配方营养油中不饱和脂肪酸的氧化酸败和提高其消化吸收,该研究以调和油为油相、乳清蛋白为壁材,将乳化剂溶于油相,超声法制备成纳米级乳化液,利用喷雾干燥法将纳米乳液制备成微胶囊粉末。以乳液粒径为响应值,用响应面分析法研究乳化剂添加量、超声功率和超声时间等因素对乳液粒径和喷雾干燥产品表面含油率的影响,优化制备工艺。结果表明,最佳工艺条件为:卵磷脂(PC)质量分数5.0%、超声功率660 W和超声时间20 min,乳液的平均粒径为(184.37±0.64)nm,微胶囊表面含油率为5.82%,依据此响应面模型数据可有效指导试验操作。喷雾干燥后的产品品质较好,在体外消化模拟试验中,在禁食和喂食状态下纳米乳液的游离脂肪酸释放量均高于未处理调和油。     

Preparation and Characterization of Water/Oil/Water Emulsions Stabilized by Polyglycerol Polyricinoleate and Whey Protein Isolate

ABSTRACT: In this study we tried to prepare stable water-in-oil-in-water (W/O/W) emulsions using polyglycerol polyricinoleate (PGPR) as a hydrophobic emulsifier and whey protein isolate (WPI) as a hydrophilic emulsifier. At first, water-in-oil (W/O) emulsions was prepared, and then 40 wt% of this W/O emulsion was homogenized with 60 wt% aqueous solution of different WPI contents (2, 4, and 6 wt% WPI) using a high-pressure homogenizer (14 and 22 MPa) to produce W/O/W emulsions. The mean size of final W/O/W droplets ranged from 3.3 to 9.9 μm in diameter depending on the concentrations of PGPR and WPI. It was shown that most of the W/O/W droplets were small (<5 μm) in size but a small population of large oil droplets (d > 20 μm) was also occasionally observed. W/O/W emulsions prepared at the homogenization pressure of 22 MPa had a larger mean droplet size than that prepared at 14 MPa, and showed a microstructure consisting of mainly approximately 6 to 7-μm droplets. When a water-soluble dye PTSA as a model ingredient was loaded in the inner water phase, all W/O/W emulsions showed a high encapsulation efficiency of the dye (>90%) in the inner water phase. Even after 2 wk of storage, >90% of the encapsulated dye still remained in the inner water phase; however, severe droplet aggregation was observed at relatively high PGPR and WPI concentrations.