Small extracellular vesicles' enrichment from biological fluids using an acoustic trap.

Small extracellular vesicles (sEVs), a form of extracellular vesicles, are lipid bilayered structures released by all cells. Large-scale studies on sEVs from clinical samples are necessary, but a major obstacle is the lack of rapid, reproducible, efficient, and low-cost methods to enrich sEVs. Acoustic microfluidics have the advantage of being label-free and biocompatible, which have been reported to successfully enrich sEVs. In this paper, we present a highly efficient acoustic microfluidic trap that can offer low and large volume compatible ways of enriching sEVs from biological fluids by flexible structure design. It uses the idea of pre-loading larger seed particles in the acoustic trap to enable sub-micron particle capturing. The microfluidic chip is actuated using a piezoelectric plate transducer attached to a silicon-glass bonding plate with circular cavities. Each cavity works as a resonant unit, excited at the frequency of both the half wave resonance in the main plane and inverted quarter wave resonance in the depth direction, which has the ability to strongly trap seed particles at the center, thereby improving the subsequent nanoparticle capture efficiency. Mean trapping efficiencies of 35.62% and 64.27% were obtained using 60 nm and 100 nm nanobeads, respectively. By the use of this technology, we have successfully enriched sEVs from cell culture conditioned media and blood plasma at a flow rate of 10 µL min-1. The isolated sEV subpopulations are characterized by NTA and TEM, and their protein cargo is determined by WB. This acoustic trapping chip provides a rapid and robust method to enrich sEVs from biofluids with high reproducibility and sufficient quantities. Therefore, it can serve as a new tool for biological and clinical research such as cancer diagnosis and drug delivery.

Improved lipase performance by covalent immobilization of Candida antarctica lipase B on amino acid modified microcrystalline cellulose as green renewable support.

Development of immobilized lipase with excellent catalytic performance and low cost is the major challenge for large-scale industrial applications. In this study, green renewable microcrystalline cellulose (MCC) that was hydrophobically modified with D-alanine (Ala) or L-lysine (Lys) was used for immobilizing Candida antarctica lipase B (CALB). The improved catalytic properties were investigated by experimental and computational methods. CALB immobilized on MCC-Ala with higher hydrophobicity showed better catalytic activity than CALB@MCC-Lys because the increased flexibility of the lid region of CALB@MCC-Ala favored the formation of open conformation. Additionally, the low root mean square deviation and the high ß-sheet and α-helix contents of CALB@MCC-Ala indicated that the structure became more stable, leading to a significantly enhanced stability (54.80% and 90.90% relative activity at 70 °C and pH 9.0, respectively) and good reusability (48.92% activity after 5 cycles). This study provides a promising avenue to develop immobilized lipase with high catalytic properties for industry applications.

Enhancing the antioxidant properties and compatibility of protein/sodium alginate film by incorporating Zanthoxylum bungeanum essential oil Pickering emulsion.

Zanthoxylum bungeanum essential oil (ZBEO) Pickering emulsion was incorporated into rice protein (RP)/sodium alginate (SA)-based film to enhance the antioxidant activity and compatibility. With increasing ZBEO content from 2 % to 4 %, the average size of ZBEO Pickering emulsion ranged from 124.28 to 165.65 nm. The best mechanical property with a tensile strength of 14.56 MPa and hydrophobicity with a water vapor permeability of 2.11 × 10-12 g⋅cm-1⋅s-1⋅Pa-1 of emulsion film were achieved with 0.8 % ZBEO. In addition, the loss of ZBEO in the emulsion films was reduced by 11-14 %. The DPPH radical scavenging activity of emulsion film with 1.2 % ZBEO was 65.54 % in 95 % ethanol. The results of Fourier transform infrared spectroscopy and molecular dynamics simulation showed that electrostatic interactions played a leading role in film formation. Overall, ZBEO Pickering emulsion is an effective method to enhance the antioxidant activity, mechanical strength and hydrophobicity of RP/SA film.

Improved physicochemical properties and in vitro digestion of walnut oil microcapsules with soy protein isolate and highly oxidized konjac glucomannan as wall materials.

This study investigated the effect of the oxidation degrees of oxidized konjac glucomannan (OKGM) on the encapsulation efficiency (EE), physicochemical and in vitro digestive properties of soy protein isolate (SPI)-based microcapsules walnut oil using experimental and computational approaches. Microcapsules had the highest EE when the ratio of OKGM and SPI to oil was 2.5:1. With increasing the oxidation degree of OKGM, the EE of microcapsules was increased and the hygroscopicity was decreased. Molecular dynamics simulation results showed that SPI/oil/highly OKGM had relatively low binding energy (-4.03 × 106 kJ/mol) and strong electrostatic interactions, which may contribute to a higher EE and lower hygroscopicity of microcapsules, respectively. The oxidative stability of the oil was markedly improved by SPI and OKGM, and microcapsules prepared with SPI and highly OKGM had the highest in vitro digestion. This study provided theoretical support for broadening the application of microcapsules prepared with SPI and OKGM.

Development of air-assisted atomization device for the delivery of cells in viscous biological ink prepared with sodium alginate.

Skin wounds, especially large-area skin trauma, would bring great pain and even fatal risk to patients. In recent years, local autologous cell transplantation has shown great potential for wound healing and re-epithelialization. However, when the cell suspension prepared with normal saline is delivered to the wound, due to its low viscosity, it is easy to form big drops in the deposition and lose them from the wound bed, resulting in cell loss and uneven coverage. Here, we developed a novel air-assisted atomization device (AAAD). Under proper atomization parameters, 1% (w/v) sodium alginate (SA) solution carrier could be sprayed uniformly. Compared with normal saline, the run-off of the SA on the surface of porcine skin was greatly reduced. In theory, the spray height of AAAD could be set to achieve the adjustment of a large spray area of 1-12 cm2. In the measurement of droplet velocity and HaCaT cell viability, the spray height of AAAD would affect the droplet settling velocity and then the cell delivery survival rate (CSR). Compared with the spray height of 50 mm, the CSR of 100 mm was significantly higher and could reach 91.09% ± 1.82% (92.82% ± 2.15% in control). For bio-ink prepared with 1% (w/v) SA, the viability remained the same during the 72-h incubation. Overall, the novel AAAD uniformly atomized bio-ink with high viscosity and maintained the viability and proliferation rate during the delivery of living cells. Therefore, AAAD has great potential in cell transplantation therapy, especially for large-area or irregular skin wounds.

Performance of nitrification-denitrification and denitrifying phosphorus removal driven by in-situ generated biogenic manganese oxides in a moving bed biofilm reactor.

Simultaneous removal of NH4+-N, NO3--N, COD, and P by manganese redox cycling in nutrient wastewater was established with two moving bed biofilm reactors (MBBRs) with in-situ generated biogenic manganese oxides (BioMnOx) and non-BioMnOx. In-situ generated BioMnOx preferentially promoted the denitrification, and the average removal of NO3--N, NH4+-N, and TN in the experimental MBBR with BioMnOx increased to 89.00%, 70.64%, and 76.06% compared with the control MBBR with non-BioMnOx. The relevant enzymes activity, extracellular polymeric substance (EPS), electron transport system activity (ETSA), and reactive oxygen species (ROS) were investigated. The element valence and morphology of purified BioMnOx were characterized by X-ray photoelectron spectroscopy (XPS) and transmission electron microscopy (TEM), as well as the effect of BioMnOx on nitrogen and phosphorus removal. The results suggested that BioMnOx could improve nitrogen conversion. Electrochemical characteristic and microbial community were detected. This study provided a new strategy for nutrients removal in BioMnOx-mediated wastewater treatment.

Interaction, bioaccessibility and stability of bovine serum albumin-gamma-oryzanol complex: Spectroscopic and computational approaches.

The study explored the interaction mechanism between bovine serum albumin (BSA) and gamma-oryzanol (GO) by spectroscopic and computational approaches and the potential to enhance bioaccessibility and chemical stability of GO in the complex with BSA. Fluorescence spectroscopy showed that GO was bound to BSA with static quenching at a single binding site, being consistent with molecular docking results. Thermodynamic analysis and molecular dynamics simulation showed that electrostatic forces dominated interactions between BSA and GO. Besides, BSA-GO complex was more stable at pH 7.4 than at pH 2.0, with low root-mean-square deviation (2.57 Å vs 12.37 Å) and low binding energy (-424.23 kJ/mol at 277 K vs -188.55 kJ/mol at 277 K), but complex stability significantly decreased with increasing temperature. The bioaccessibility and stability of GO in the complex were significantly higher than those in water. This study provided theoretical support for developing proteins as delivery system for GO.

Improved catalytic properties of Candida antarctica lipase B immobilized on cetyl chloroformate-modified cellulose nanocrystals.

The catalytic activity of Candida antarctica lipase B (CALB) immobilized on modified cellulose nanocrystals (CNC) with different hydrophobicity was investigated using experimental and theoretical approaches. Firstly, the modified CNC were characterized by multi-spectroscopic methods, water contact angle, scanning electron microscopy and thermogravimetric analysis. Moderately hydrophobic CNC were found to be an optimal support for CALB immobilization. Secondly, model systems contained a CALB molecule and different numbers of modified CNC molecules (CALB@3CNC-C16, CALB@10CNC-C16 and CALB@15CNC-C16) were prepared for molecular dynamics (MD) simulation. Root-mean-square fluctuation values (0.61-2.61 Å) of lid region were relatively high in CALB@10CNC-C16, indicating that modified CNC with moderate hydrophobicity favored forming a lid-open conformation of CALB. Finally, the esterification of oleic acid catalyzed by the immobilized CALB showed higher conversion (54.68 %) than free CALB (12.98 %). Insights into modified CNC with tunable properties provided by this study may be a potential support for improving the catalytic performance of lipases.

Performance of simultaneous nitrification-denitrification and denitrifying phosphorus and manganese removal by driving a single-stage moving bed biofilm reactor based on manganese redox cycling.

Simultaneous removal of NH4+-N, NO3--N, COD, and P by manganese redox cycling in nutrient wastewater was established with a single-stage moving bed biofilm reactor (MBBR) under low C/N ratio. When sodium succinate replaced the conventional denitrifying carbon source, removal efficiencies of TN, NO3--N, NH4+-N, TP, and Mn2+ were 65.13 %, 79.63 %, 92.79 %, 51.57 %, and 68.10 %, respectively. Based on modified Stover-Kincannon model, 11.03 and 10.05 mg TN·L-1·h-1 of Umax values were obtained with sodium acetate and sodium succinate as substrates. Extracellular polymeric substances were used to evaluate the characteristics of biofilm, and microbial community of biofilm was identified. Transformation processes of NO3--N, NH4+-N, Mn2+, and P were investigated, suggesting that the main functional groups (e.g., CO, Mn-O, and CN bonds) participated in N, P, and Mn2+ removal, and MnO2 was the main component of biogenic manganese oxides. This study provides a new strategy for nutrients removal by Mn2+ driven MBBR.

Host-guest interactions between oleic acid and ß-cyclodextrin: A combined experimental and theoretical study.

The physicochemical properties and interactions of the complex of oleic acid (OA) and ß-cyclodextrin (ß-CD) were studied by experimental and computational methods. Differential scanning calorimetry and X-ray diffraction confirmed the successful preparation of OA/ß-CD inclusion complex. The stability of the complex was improved, exhibiting a 61.2 °C higher degradation temperature and a lower peroxide value than OA. Raman and Fourier transform infrared spectroscopy studies revealed that the carboxyl group of OA entered into the ß-CD cavity to form hydrogen bonds, which was confirmed by conformational search and weak interactions analysis. Dispersion energy from van der Waals (-290.79 kJ/mol) contributed 87.3% to total interaction energy (-253.88 kJ/mol). Topological analysis showed that four moderate hydrogen bonds were formed between OA and ß-CD with the bond energy ranging from -76.05 to -30.25 kJ/mol. This work provided theoretical basis for the development of nutritional supplements containing unsaturated fatty acids encapsulated by ß-CD.

Performance and mechanism of simultaneous nitrification-denitrification and denitrifying phosphorus removal in long-term moving bed biofilm reactor (MBBR).

The long-term moving bed biofilm reactor (MBBR) with carrier-attached biofilm was successfully operated for simultaneous removal of nitrogen, phosphorus, and COD at various C/N ratios. Results indicated that 99.60%, 63.58%, 78.94%, and 59.64% of NH4+-N, NO3--N, TN, and TP were removed at C/N ratio, hydraulic retention time (HRT), and carrier film amount of 5, 40 h, and 1.2 mg·g-1. Nitrogen balance analysis showed that more than 89% of nitrogen (C/N = 20, 15, 10, 5) was converted to gas products. Extracellular polymeric substances (EPS), electron transport system activity (ETSA), and enzyme activity of biofilm were evaluated. Protein (PN)/polysaccharose (PS) values and ETSA decreased with the decrease of C/N ratios. Metagenomics sequencing further revealed that the prominent phyla for nitrogen and phosphorus removal were identified including Proteobacteria, Acidobacteria, Nitrospirae, and Chloroflexi. Proteobacteriaand Gammaproteobacteria were identified as the dominant denitrifying phosphate accumulating organisms (PAO) at the phylum and class level, respectively.

Sheathless acoustic based flow cell sorter for enrichment of rare cells.

Cell enrichment is a powerful tool in many kinds of cell research, especially in applications with low abundance cell types. In this work, we developed a microfluidic fluorescence activated cell sorting device that was able to perform on-demand, low loss cell detection, and sorting. The chip utilizes three-dimensional acoustic standing waves to position all cells in the same fluid velocity regime without sheath. When the cells pass through a laser interrogation region, the scattering and fluorescent signals are detected, translated and transported to software. The target cells are then identified by gating on the plots. Short bursts of standing acoustic waves are triggered by order from PC to sort target cells within predefined gating region. For very low abundance and rare labeled lymphocytes mixed with high concentration unlabeled white blood cells (WBCs), (1-100 labeled lymphocytes are diluted in 106 WBCs in 1 ml volume fluid), the device is able to remove more than 98% WBCs and recover labeled lymphocytes with efficiency of 80%. We further demonstrated that this device worked with real clinical samples by successfully isolating fetal nucleated red blood cells (FNRBCs) in the blood samples from pregnant women with male fetus. The obtained cells were sequenced and the expressions of (sex determining region Y) SRY genes were tested to determine fetal cell proportion. In genetic analysis, the proportion of fetal cells in the final picked sample is up to 40.64%. With this ability, the device proposed could be valuable for biomedical applications involving fetal cells, circulating tumor cells, and stem cells.

Potential Application of Lonicera japonica Extracts in Animal Production: From the Perspective of Intestinal Health.

Lonicera japonica (L. japonica) extract is rich in active substances, such as phenolic acids, essential oils, flavones, saponins, and iridoids, which have a broad spectrum of antioxidant, anti-inflammatory, and anti-microbial effect. Previous studies have demonstrated that L. japonica has a good regulatory effect on animal intestinal health, which can be used as a potential antibiotic substitute product. However, previous studies about intestinal health regulation mainly focus on experimental animals or cells, like mice, rats, HMC-1 Cells, and RAW 264.7 cells. In this review, the intestinal health benefits including antioxidant, anti-inflammatory, and antimicrobial activity, and its potential application in animal production were summarized. Through this review, we can see that the effects and mechanism of L. japonica extract on intestinal health regulation of farm and aquatic animals are still rare and unclear. Further studies could focus on the regulatory mechanism of L. japonica extract on intestinal health especially the protective effects of L. japonica extract on oxidative injury, inflammation, and regulation of intestinal flora in farm animals and aquatic animals, thereby providing references for the rational utilization and application of L. japonica and its extracts in animal production.

Interaction between Curcumin and ß-Casein: Multi-Spectroscopic and Molecular Dynamics Simulation Methods.

Effect of temperature and pH on the interaction of curcumin with ß-casein was explored by fluorescence spectroscopy, ultraviolet-visible spectroscopy and molecular dynamics simulation. The spectroscopic results showed that curcumin could bind to ß-casein to form a complex which was driven mainly by electrostatic interaction. The intrinsic fluorescence of ß-casein was quenched by curcumin through static quenching mechanism. The binding constants of curcumin to ß-casein were 6.48 × 104 L/mol (298 K), 6.17 × 104 L/mol (305 K) and 5.73 × 104 L/mol (312 K) at pH 2.0, which was greater than that (3.98 × 104 L/mol at 298 K, 3.90 × 104 L/mol at 305 K and 3.41 × 104 L/mol at 312 K) at pH 7.4. Molecular docking study showed that binding energy of ß-casein-curcumin complex at pH 2.0 (-7.53 kcal/mol) was lower than that at pH 7.4 (-7.01 kcal/mol). The molecular dynamics simulation study showed that the binding energy (-131.07 kJ/mol) of ß-casein-curcumin complex was relatively low at pH 2.0 and 298 K. α-Helix content in ß-casein was decreased and random coil content was increased in the presence of curcumin. These results can promote a deep understanding of interaction between curcumin and ß-casein and provide a reference for improving the bioavailability of curcumin.

A mutant T1 lipase homology modeling, and its molecular docking and molecular dynamics simulation with fatty acids.

A thermostable T1 lipase from Geobacillus zalihae exhibits broad substrate specificity and good potential application in fats and oils. However, structural insight into the enzyme against substrates is poorly understood at the molecular level. Herein, the study aimed to examine interactions between a mutant T1 lipase (Mut-T1 lipase) and selected fatty acids (caprylic, myristic, stearic, oleic, linoleic and linolenic acids) by performing molecular docking and molecular dynamics (MD) simulation. The structure of Mut-T1 lipase obtained by homology modeling was reliable for molecular docking and MD simulation. Molecular docking revealed that Mut-T1 lipase showed low binding affinity for caprylic acid (-4.97 kcal/mol) compared to the other fatty acids (-5.65 to -6.88 kcal/mol). However, the conformation of Mut-T1 lipase-caprylic acid complex was comparably stable during the simulation, in terms of less root-mean square fluctuation. Besides, solvent accessible surface area value of Mut-T1 lipase-fatty acid complexes decreased with increasing chain length of fatty acid. van der Waals interactions were requisite in maintaining complex stability during the binding process. This work provides structural insight into interactions between the lipase and the fatty acids, which will facilitate design and applications of new mutants of T1 lipase in modifying fats and oils.

Ternary composite films with simultaneously enhanced strength and ductility: Effects of sodium alginate-gelatin weight ratio and graphene oxide content.

This study aimed to simultaneously enhance the strength and ductility of films by adding graphene oxide (GO) into the sodium alginate (SA)-gelatin film system to prepare SA-gelatin-GO ternary composite films. The effects of the SA/gelatin weight ratio and the GO content on the morphology, structural, mechanical, hydrophobic, water vapor barrier, hygroscopic, and optical properties of composite films were investigated. Results showed that tensile strength and elongation at break values of the composite film with 0.5% GO content were significantly improved from 37.00 to 48.01 MPa and 20.19% to 25.75%, respectively, compared with the SA-gelatin film without GO, suggesting GO with the content of 0.5% simultaneously enhance the strength and ductility of the SA-gelatin film, which was due to the interaction of hydrogen bonds and the good dispersibility of GO in the film matrix. The surface hydrophobicity of composite films increased first and then decreased with increasing GO content. In addition, the hygroscopicity of composite films reduced due to the GO addition, especially when the GO content was 0.25%. The SA-gelatin-GO ternary composite films provide a good select for food packaging materials, especially the materials that require high strength and fine ductility.

Improving the Physical and Oxidative Stability of Emulsions Using Mixed Emulsifiers: Casein-Octenyl Succinic Anhydride Modified Starch Combinations.

This study aims to investigate the influence of casein and octenyl succinic anhydride modified starch (OSAS) combinations on the physical and oxidative stability of fish oil-in-water emulsions. The interaction between casein and OSAS was manifested in changes in protein structure and hydrogen-bonding interaction. Casein-OSAS combinations could effectively inhibit droplet aggregation at pH 4 and attenuate droplet growth at a high CaCl2 concentration of 0.2 mol/L, compared with casein as an emulsifier. Nanoemulsions stabilized by casein-OSAS combinations or casein showed better oxidative stability compared with OSAS-stabilized emulsions. Therefore, casein-OSAS combinations can improve some physical properties of protein-based emulsions and oxidative stability of modified starch-based emulsions, suggesting protein-modified starch combinations are more promising in the emulsion-based food industry compared to each of the two emulsifiers alone.

Improving the stability of phosphatidylcholine-enhanced nanoemulsions using octenyl succinic anhydride-modified starch.

Phosphatidylcholine-coated emulsions are unstable and prone to phase separation under certain environmental stresses, such as low pH and moderate ionic strength (>100 mmol/L NaCl). This study investigated the formation and stability of octenyl succinic anhydride-modified starch (OSAS)-stabilized nanoemulsions enhanced with phosphatidylcholine for the encapsulation of soybean oil. The effect of variables (OSAS/phosphatidylcholine weight ratio, oil composition, emulsifier concentration, etc.) and environmental stresses (pH, temperature, ionic strength, etc.) on the stability of phosphatidylcholine-enhanced nanoemulsions stabilized by OSAS was examined. Adequate addition of OSAS (OSAS/phosphatidylcholine weight ratio = 3:7) led to the formation of a more stable nanoemulsion. An emulsion at pH 2 was unstable against creaming, and no phase separation occurred after 130 h; presumably, OSAS has a certain degree of steric repulsion that protects the nanoemulsion from oiling-off. The phosphatidylcholine-enhanced nanoemulsions stabilized by OSAS were stable at high NaCl concentrations (≤800 mmol/L) and temperatures from 30 to 90 °C, which was attributed to steric and electrostatic repulsions. Transmission electron microscopy images showed that OSAS and phosphatidylcholine were both adsorbed around the surface of the emulsion. These results showed that adding OSAS could improve the formation and stability of phosphatidylcholine-stabilized emulsions.

Effects of an immune stimulant, inactivated Mycobacterium phlei, on the growth performance as well as meat quality of fattening pigs.

Inactivated mycobacterium phlei (M. phlei) is well known for its immune-stimulatory functions in humans and livestock, but less information is available about the influence on meat quality of pigs when used as a feed additive. This study was designed to evaluate the effects of inactivated M. phlei on growth performance as well as meat quality of fattening pigs. A total of 240 cross-bred pigs ([Landrace × Yorkshire] × Duroc) with initial body weight of 80.14 ± 0.29 kg were randomly allocated to five treatments, each of which consisted of eight replicates with 6six pigs per replicate. The basal diet supplemented with five levels of inactivated M. phlei preparations (0, 3.5 × 109 [0.1% w/w], 7 × 109 [0.2%], 1.4 × 1010 [0.4%] or 2.1 × 1010 [0.6%] colony-forming units/kg) was respectively fed to the control group and four treatment groups for 30 days. Adding 0.4% of inactivated M. phlei to diet increased the average daily feed intake and average daily gain of pigs. Importantly, intramuscular fat percentage in the Longissimus dorsi (LD) was increased by feeding diet containing 0.2%, 0.4% and 0.6% of inactivated M. phlei, despite the pH value, drip loss, cooking loss and filter paper fluid uptake not being influenced. Analysis of the fatty acid components showed that some saturated fatty acids were decreased in LD after feeding inactivated M. phlei, but some monounsaturated fat acids (MUFAs) and polyunsaturated fatty acids were increased (PUFAs), which induced the total contents of MUFAs and PUFAs were improved. RT-PCR assay revealed that feeding inactivated M. phlei up-regulated genes implicated in fat metabolism in muscle, including ELOVL6, FASN, SCD1 and H-FABP. This study revealed that feeding inactivated M. phlei not only increased growth performance of fattening pigs, but also improved the meat quality by increasing intramuscular fat content, thus inactivated M. phlei probably has high utilization value in modern pig production.

Improved Physicochemical Properties of Yogurt Fortified with Fish Oil/γ-Oryzanol by Nanoemulsion Technology.

Fish oil has several dietary benefits, but its application in food formulations is limited because of its poor water-solubility, easy oxidation and strong odor. The purposes of this study were to produce a fish oil/γ-oryzanol nanoemulsion and to evaluate the effect of adding this nanoemulsion on the physicochemical and sensory characteristics of yogurts. Adding fish oil/γ-oryzanol nanoemulsion resulted in a significant reduction in the acidity and syneresis of yogurt. Yogurt with the nanoemulsion had significantly lower peroxide value (0.28 mmol/L after 21 days) and higher retention of eicosapentaenoic acid and docosahexaenoic acid contents (decreased to 95% and 94% of its initial value, respectively) than yogurt with fish oil/γ-oryzanol (peroxide value = 0.65 mmol/L; eicosapentaenoic acid and docosahexaenoic acid contents decreased to 72% and 53% of its initial value, respectively). Fish oil/γ-oryzanol nanoemulsion incorporated into yogurt had closer sensory attributes scores to plain yogurt. This study may have important implications for the application of fish oil/γ-oryzanol nanoemulsion in yogurt.