Rheology and stability of oil-in-water emulsions stabilised with african catfish mucilage

Abstract

The mucilage secreted from African Catfish (Clarias gariepinus) when fish is stressed is a waste product that has lubricating properties and contains polysaccharides, proteins, and nucleic acid. African catfish mucilage (ACM) could be a potential emulsifier, however, information on its emulsification properties is scarce. Emulsions are thermodynamically unstable and could break down over time. Demand for sustainable natural emulsifiers has been documented in the literature and a number of them are used as food ingredients. Hence, my overall original contribution to knowledge is to characterise the physicochemical, stability, and rheological properties of ACM, a waste from African catfish to provide an effective suitable, eco-friendly alternative emulsifier that could stabilise food-grade, cosmetics, drug delivery, and personal care products. The stability of freeze-dried ACM in MilliQ water was measured with a zetasizer and Turbiscan MA 2000. The functional groups in ACM were determined using Attenuated Total Reflection (ATR)-Fourier Transform Infrared (FTIR) spectroscopy while the flow and viscoelastic behavior of ACM was measured using a rheometer. ACM is a stable material with negatively charged (-38mV) loosely bound electrons having polar and non-polar portions. Turbiscan revealed that ACM in MilliQ water was stable after 180 minutes of storage. Wavelength peaks for ACM in the range of 400-4000 cm-1 showed that it contained amphiphilic protein as functional groups which is an essential characteristic for stability. ACM showed shear-thinning behavior when subjected to shear rates of 1, 10, and 100 s 1. ACM was found to be a pseudoplastic, non-Newtonian, and viscoelastic material with higher G’ and lower G’’ with increasing deformation at a fixed angular frequency of ω = 1 rad/s. The moduli in the linear viscosity range (LVR) was constant at low strains of up to 0.2 % and had cross-over points at about 10 %. Although ACM had a short LVR, rheological stability was conferred through the amphiphilic filamentous protein threads in ACM. Also, ACM could form strong interactive hydrogen and covalent bonds (emulsifying ability) with materials that have both polar and non-polar phases. The Casson model gave the best fit for African catfish mucilage. A Zetasizer and Turbiscan were used to measure stability properties of ACM on soy milk, morphology was determined with Transmission electron microscopy (TEM), while functional groups in ACM and ACM-stabilised soya milk emulsions were determined using ATR-FTIR. The ATR-FTIR spectra of stabilised emulsion revealed synergies with soya milk droplets. Turbiscan revealed that ACM concentrations of 1, 3, and 5 % w/w stabilised soy milk emulsions within 180 min of storage. The particle size was determined with the zetasizer and stability was expressed as a function of Sauter mean diameter (D₍₃‚₂₎) calculated from the particle size. The higher the concentration of ACM, the more stable the emulsion i.e. the lower the D₍₃‚₂₎ of stabilised emulsions. Also, the higher the ACM-emulsifier soy milk ratio (ESMR) the higher the stability i.e. the lower the D₍₃‚₂₎ of stabilised emulsions. The ESMR influenced the stability of ACM stabilised soy milk emulsions based on BS % flux and the D (3,2) values. The trend was that the higher ESMR the higher the BS % flux and the lower the D (3,2) values. The spectra of ACM stabilised soy milk emulsion revealed interactions between ACM and soya milk droplets. The results from TEM, ATR-FTIR, particle size, and BS % flux analysis showed that the mucins in ACM formed strong cohesive connections with stabilised soy milk emulsions and the ACM exhibited adhesive properties. Two-way analysis of variance (ANOVA) after Tukey’s multiple comparisons (TMC) test established a significant difference (𝜌 < 0.05) on ACM stabilised soy milk emulsions with ESMRs of 5:10, 5:30, and % 5:50 %. ANOVA studies show that both BS % flux which closely fit the regression line by 99.94% and D₍₃‚₂₎ which had a 90% fit were good predictors of the stability of these emulsions. Distinct physicochemical properties like functional groups, morphology, Sauter mean diameter ((D(3,2)), and backscattering (BS) % flux were measured to determine nanoemulsion stability using ultrasonication. The emulsifying ability of ACM on O/W-type nanoemulsions was investigated by D-optical mixture design methodology. The D-optical mixture design was used in preparing the ACM stabilised O/W-type nanoemulsion using ultrasonic cavitation. Turbiscan analysis confirmed the backscattering profile and Cryo-TEM studies confirmed the structural stability of the nanoemulsions. The ATR FTIR spectra revealed that the amphiphilic proteins in ACM formed interfaces with oil and water simultaneously to confer stability on ACM stabilised nanoemulsions which is consistent with micelle theory. The emulsifier-oil ratio (EOR) influenced the stability of O/W-type nanoemulsion based on BS % flux and the D (3,2). The trend was that the higher EOR the higher the stability i.e. the higher the BS % flux and the lower the D (3,2) of O/W-type nanoemulsion. In this study, the D-Optimal mixture design was used in optimising O/W–type nanoemulsion formed by ultrasonication from sunflower oil, MilliQ water, and African catfish mucilage. Three mixture components constrained at lower and upper limits (ACM: 1 to 5 %, oil 3 to 10 %, and water: 85 to 96 %) were evaluated for their effects on responses of BS % flux and D(3,2). A quadratic mixture model was the most appropriate for both BS % flux and D(3,2). The quadratic mixture model for BS % flux was significant (F [557.86, 8.88] = 62.80; p-value < 0.0001). The model’s lack of fit (F [19.98, 6.990E-003] = 2858.24; p < 0.0001) was also significant, however, the predicted R-squared value was 0.9123 and adequate precision was 20.186 indicating a model with adequate goodness-of-fit to the experimental data. Similarly, the quadratic mixture model for D(3,2) was significant (F [235.71, 0.13] = 1805.99; p-value < 0.0001). The model lack of fit (F [0.17, 0.11] = 0.3104; p = 1.49) was not significant, and the predicted R-squared value was 0.9977 and adequate precision was 104.158 indicating a model with adequate goodness-of-fit. Hence both BS % flux and the D (3,2) were good predictors of stability of O/W-type nanoemulsions. Desirability functions were chosen to either maximise BS % flux (𝛾1) and minimise Sauter mean diameter (𝛾2) or maximise BS % flux (𝛾1) and maximise Sauter mean diameter (𝛾2). Overall, positive physicochemical and functional properties were observed for ACM as it was established that it could stabilise O/W-type nanoemulsions, and it was shown that an optimal ACM stabilised O/W-type nanoemulsions could be produced. The flow and viscoelastic properties were investigated to assess the stability and quality of ACMs’ most stable emulsions and nanoemulsions based on rheological data. All the ACMs’ most stable emulsions and nanoemulsions and control soya milk showed shear-thinning behavior as viscosity decreased as shear rate was increased from 0.01 to 1000 s -1. The non-Newtonian flow of soy milk (control), ACM stabilised soya milk emulsion, ACM stabilised O/W-type nanoemulsion, and ACM stabilised O/W-type nanoemulsion enriched with soy milk fiber, was modeled with Power, Herschel-Buckley, Casson, and Bingham law equations, and the Casson model was observed to be the best fit. The moduli in the linear viscoelastic region (LVR) of ACM stabilised O/W-type nanoemulsions, and ACM stabilised O/W-type nanoemulsions enriched with soya milk fiber was constant at low strains of 0.4 and 0.6 % and cross-over points of about 30 and 50 % respectively at a fixed angular frequency of ω = 1 rad/s. The moduli in the LVR indicated improved stability for ACM stabilised O/W-type nanoemulsion enriched with soy milk fiber in comparison to that of ACM stabilised O/W-type nanoemulsion. This was due to the synergy between ACM and soy milk fiber which improved the moduli of ACM stabilised O/W-type nanoemulsion as the moduli did not match-up to the simple addition of each modulus. This synergistic effect enhanced the web-like stable matrix structure of the mixture as a phase change was not observed. All ACM stabilised O/W-type nanoemulsions formed a stable network structure as phase separation was not observed within the frequency range of 1.0 to 100 rad/s. At low frequencies, the elastic portion dominated the curves as the storage modulus was always higher than the loss modulus. Both ACM stabilised O/W-type nanoemulsion and ACM stabilised O/W-type nanoemulsion enriched with fiber behaved as weak gels. Refrigerating i.e. keeping at temperatures of 5°C sustained and improved the structural stability and viscoelastic rheological properties of the ACM, ACM stabilised O/W-type nanoemulsion and ACM stabilised O/W-type nanoemulsions enriched with soya milk fiber. An increase in time duration did not have significant negative effects on the structural stability and viscoelastic properties of the ACM, ACM stabilised O/W-type nanoemulsion and ACM stabilised O/W-type nanoemulsions enriched with soya milk fiber as ACM and all its most stable nanoemulsions remained stable with time. This was due to the filamentous proteins of ACM which was responsible for the cross-linkage stable structure network of ACM and ACMs’ most stable nanoemulsions. Consequently, the results from morphology, BS % flux, D(3,2) (particle size), spectral functional groups and mucoadhesive property of ACM correlated with rheological properties as the stability and viscoelastic properties of ACM stabilised O/W-type nanoemulsions were ascribed to the amphiphilic filamentous protein threads present in the African catfish mucilage. These results show that African catfish mucilage is an effective suitable, an eco-friendly alternative emulsifier that could serve as feedstock in food-grade, cosmetics, drug delivery, and personal care industry.