Optimized cultural conditions of functional yogurt for γ-aminobutyric acid augmentation using response surface methodology

Yogurt, a functional dairy food product, is an effective medium for delivering beneficial functional ingredients. One ingredient, γ-aminobutyric acid (GABA), has growing appeal in the development of functional foods for its potential in reducing the risk of diabetes, hypertension, and stress as a bioactive agent. However, the concentration of GABA in existing food products is remarkably low. We developed a functional yogurt rich in GABA using Streptococcus thermophilus fmb5. The GABA yield of yogurt was enhanced by optimization of culture conditions using single factor and response surface methods. The results showed that culture temperature, monosodium glutamate concentration, and culture time are the 3 main factors that affect GABA yield. The optimal culture conditions were determined as follows: 38.8°C for culture temperature, 20 g/L of monosodium glutamate, and 120 h of culture time. Under the above optimal conditions, the actual yield of GABA production was maximized at 9.66 g/L, which was 1.2 times or higher than that of from any single factor treatment. The GABA concentration, viable bacteria number, and water-holding capacity of GABA-rich yogurt were stable throughout the whole storage time. The results show that producing yogurt with Streptococcus thermophilus fmb5 and the optimized culture conditions will achieve high GABA concentrations that maximize health benefits to consumers.     

Optimisation of immobilisation conditions for chick pea β-galactosidase (CpGAL) to alkylamine glass using response surface methodology and its applications in lactose hydrolysis

Response surface methodology was advantageously used to optimally immobilise a β-galactosidase from chick pea onto alkylamine glass using Box–Behnken experimental design, resulting in an overall 91% immobilisation efficiency. Analysis of variance was performed to determine the adequacy and significance of the quadratic model. Immobilised enzyme showed a shift in the optimum pH; however, optimum temperature remained unaffected. Thermal denaturation kinetics demonstrated significant improvement in thermal stability of the enzyme after immobilisation. Galactose competitively inhibits the enzyme in both soluble and immobilised conditions. Lactose in milk whey was hydrolysed at comparatively higher rate than that of milk. Immobilised enzyme showed excellent reusability with retention of more than 82% enzymatic activity after 15 uses. The immobilised enzyme was found to be fairly stable in both dry and wet conditions for three months with retention of more than 80% residual activity.     

Amyloglucosidase-catalyzed synthesis of n-octyl-d-glucoside—analysis using response surface methodology

Amyloglucosidase (3.2.1.3)-catalyzed synthesis of n-octyl-d-glucoside was optimized using response surface methodology. A central composite rotatable design involving 32 experiments of five variables at five levels was employed to study the glucosylation reaction. Among the variables studied, namely, n-octanol (15–75 MEq to d-glucose), enzyme (20–100 mg ), pH (4.0–8.0), buffer volume (0.2–1.0 ml) and temperature (30–70°C), amyloglucosidase concentration, pH and temperature were found to be significant. Experimental data fitted the second-order polynomial equation well, as indicated by an R² value of 0.895. Validation experiments carried out under predicted conditions showed good correspondence between experimental and predicted yields. Various surface plots were generated to describe the relationship between operating variables and the conversion yields. The highest yield of 53.5% predicted at optimum conditions of 75 Eq n-octanol, 20 mg amyloglucosidase, 0.2 ml, pH 7.8 buffer at 50 °C showed good correspondence to the experimental yield of 53.8% under these conditions.     

Characterization of β-carotene nanoemulsions prepared by microfluidization technique

In the present work, β-carotene nanoemulsions, as potential active ingredients for liquid food, were prepared using high pressure homogenization. The influence of different homogenizing conditions (pressure and number of cycles) and emulsifier type and concentrations on particle size parameters and content of β-carotene was investigated. The droplet size of the emulsions was found to decrease from 416.0 to 97.2 nm with increasing microfluidization pressure, number of cycles, and emulsifier concentration. The optimum conditions for preparing β-carotene nanoemulsions were determined to be homogenization pressure of 120 MPa and 3 cycles. The storage study showed that the nanoemulsions were physically stable for about 5 weeks at room temperature (25°C). β-Carotene degradation was considerably slower in WPI-stabilized nanoemulsions than in Tween 20-stabilized ones, which was attributed to the increased surface area. These results have important consequences for the design and utilization of food-grade nanoemulsions.     

Optimization of medium constituents for ε-poly-L-lysine fermentation with response surface methodology

Abstract: ɛ-Poly-L-lysine (ɛ-PL) is a novel food biopreservative with broad antimicrobial activity. In the present study, single-factor experiments were applied to screen optimal carbon and nitrogen sources that affect the yield of a ɛ-PL-producing strain, Streptomyces violaceusniger, which was obtained in our laboratory recently. Box-Behnken design and response surface methodology (RSM) were adopted to derive a statistical model for optimizing the composition of the fermentation medium. The results showed that the main factors that affect ɛ-PL production were glucose, carbon source; peptone, organic nitrogen source; and (NH₄)₂SO₄, inorganic nitrogen source. The optimum fermentation medium for ɛ-PL production of S. violaceusniger consisted of (g/L) glucose, 62.49; peptone, 5.71; (NH₄)₂SO₄, 11.19; MgSO₄, 0.5; FeSO₄, 0.03; ZnSO₄, 0.04; KH₂PO₄, 1.36; and Na₂HPO₄.12H₂O, 3.58. When cultured in the optimum medium, the ɛ-PL production was an average of 0.349 ± 0.025 mg/mL, which was 1.75 times higher than the initial medium. Practical Application: The present study illustrated the potential use of response surface methodology with the Box-Behnken design in biotechnology. Results from this work suggested that ɛ-PL production was increased greatly with the optimal medium constituents by the potential organism, S. violaceusniger, which may be a good resource for the industrial production of ɛ-PL in future.     

Rheological and textural studies of fresh and freeze-thawed native sago starch–sugar gels. I. Optimisation using response surface methodology

A three-factor–three-level Box–Behnken design was adopted to study the simultaneous effects of two compositional variables (6–8% sago starch and 25–35% sugar) and one processing variable (shearing speed of mixer at 20–50 rpm) on textural and rheological properties of gels. Analysis of variance (ANOVA) was performed to evaluate the potential interactive and quadratic effects between these variables. Sago starch and sugar levels both increased gel stiffness and viscoelasticity. Shearing, on the other hand, reduced gel stiffness and viscoelasticity. Ridge analysis was performed to estimate the values of these variables which maximised and minimised the textural parameters of hardness, gumminess, resilience, cohesiveness, and springiness. Pearson correlations among various rheological and textural properties of gels were studied. The processing conditions that contributed to an optimum gel setting were found at sago starch of level of 7.69%, sugar of 30.29%, and shearing speed of 45.86 rpm.     

Performance of selected emulsifiers and their combinations in the preparation of β-carotene nanodispersions

In this work, the characteristics of β-carotene nanodispersions prepared with sodium caseinate (SC), Tween 20, decaglycerol monolaurate (ML750) and sucrose fatty acid ester (L1695) using solvent displacement technique were compared. The mean particle size of the nanodispersions ranged from 30 nm to 206 nm, depending on the emulsifier used. SC-stabilized nanodispersions had the largest particles size, while those prepared with Tween 20 had the smallest. The mean particle size and size distribution of the nanodispersions generally remained unchanged after 8 weeks of storage time at 4 °C, but the β-carotene content in all nanodispersions decreased over time. The β-carotene in SC-stabilized nanodispersions was the most stable against oxidation among all samples, most likely due to the small specific surface area of the particles, physical barrier of SC protecting the encapsulated β-carotene against free radicals and the antioxidative activity of caseins. The combined effect of SC and ML750 or L1695 on the mean particle size and β-carotene retention was also investigated. Nanodispersions prepared with SC-ML750 or SC-L1695 showed larger mean particle sizes compared to those prepared with ML750 or L1695 alone. However, the stability of β-carotene in these systems was improved owing to the antioxidative activity of SC. This work demonstrated the importance of emulsifier in determining the characteristics and stability of β-carotene nanodispersions.     

Optimisation of the formulation of β-carotene loaded nanostructured lipid carriers prepared by solvent diffusion method

In this study, the optimised β-carotene loaded nanostructured lipid carriers (NLC) were prepared using the solvent diffusion method. Response surface methodology (RSM) was employed in conjunction with a central composite design (CCD) to evaluate the effect of the preparation variables on particle size and β-carotene stability to optimise the NLC formulation. Quadratic polynomial was the best fitted mathematical model for the experimental results. The statistical evaluations revealed that the lipid phase concentration and the surfactant concentration had significant effect on particle size of NLC. In addition, the influence of the liquid lipid to total lipid ratio and temperature on β-carotene degradation was more important. The optimum formulations with minimum particle size (8–15 nm) and low β-carotene degradation (0–3%) were derived from the fitted models and were experimentally examined which demonstrated a reasonable agreement between experimental and predicted values. Transition electron microscopy (TEM) observations exhibited spherical morphology of β-carotene loaded NLC.     

Optimization of β-casein stabilized nanoemulsions using experimental mixture design

The objective of this study was to determine the effect of changing viscosity and glass transition temperature in the continuous phase of nanoemulsion systems on subsequent stability. Formulations comprising of β-casein (2.5%, 5%, 7.5%, and 10% w/w), lactose (0% to 20% w/w), and trehalose (0% to 20% w/w) were generated from Design of Experiments (DOE) software and tested for glass transition temperature and onset of ice-melting temperature in maximally freeze-concentrated state (T(g) ' & T(m) '), and viscosity (μ). Increasing β-casein content resulted in significant (P < 0.0001) increases in viscosity and T(m) ' (P= 0.0003), and significant (P < 0.0001) decreases in T(g) '. A mixture design was used to predict the optimum levels of lactose and trehalose required to attain the minimum and maximum T(g) ' and viscosity in solution at fixed protein contents. These mixtures were used to form the continuous phase of β-casein stabilized nanoemulsions (10% w/w sunflower oil) prepared by microfluidization at 70 MPa. Nanoemulsions were analyzed for T(g) ' & T(m) ', as well as viscosity, mean particle size, and stability. Increasing levels of β-casein (2.5% to 10% w/w) resulted in a significant (P < 0.0001) increase in viscosity (5 to 156 mPa.s), significant increase in particle size (P= 0.0115) from 186 to 199 nm, and significant decrease (P= 0.0001) in T(g) ' (-45 to -50 °C). Increasing the protein content resulted in a significant (P < 0.0001) increase in nanoemulsion stability. A mixture DOE was successfully used to predict glass transition and rheological properties for development of a continuous phase for use in nanoemulsions.     

Nanoemulsions of β-carotene using a high-energy emulsification-evaporation technique

Nanoemulsions of β-carotene were prepared using a high-energy emulsification-evaporation technique based on a 2³ level factorial design. Results show that it is possible to obtain dispersions at a nanoscale range. Process parameters such as time and shear rate of homogenization affected significantly particle size distribution in terms of volume-weighted mean diameter and surface-weighted mean diameter. The obtained nanoemulsions presented a volume-surface diameter ranging from 9 to 280 nm immediately after the production of particles, displaying in all cases a monomodal size distribution. Those nanoemulsions showed a good physical stability during 21 days storage. The stability was evaluated by the maintenance of size distribution. However, β-carotene retention inside the micelles and color were affected by storage. Processing conditions also influenced storage stability.     

Inhibition of β-carotene degradation in oil-in-water nanoemulsions: Influence of oil-soluble and water-soluble antioxidants

The utilisation of carotenoids as functional ingredients (pigments and nutraceuticals) in many food and beverage products is currently limited because of their poor water-solubility, high melting point, chemical instability, and low bioavailability. This study examined the impact of antioxidants on the chemical degradation of β-carotene encapsulated within nanoemulsions suitable for oral ingestion. β-Carotene was incorporated into oil-in-water nanoemulsions stabilized by either a globular protein (β-lactoglobulin) or a non-ionic surfactant (Tween 20). Nanoemulsions were then stored at neutral pH and their physical and chemical stability were monitored under accelerated stress storage conditions (55 °C). β-Carotene degradation was monitored non-destructively using colour reflectance measurements. The rate of β-carotene degradation decreased upon addition of water-soluble (EDTA and ascorbic acid) or oil-soluble (vitamin E acetate or Coenzyme Q10) antioxidants. EDTA was more effective than ascorbic acid, and Coenzyme Q10 was more effective than vitamin E acetate. The utilisation of water-soluble and oil-soluble antioxidants in combination (EDTA and vitamin E acetate) was less effective than using them individually. Emulsions stabilized by β-lactoglobulin were more stable to colour fading than those stabilized by Tween 20. These results provide useful information for designing effective nanoemulsion-based delivery systems that retard the chemical degradation of encapsulated carotenoids during long term storage.     

Optimisation of soybean hydrothermal treatment for the conversion of β-glucoside isoflavones to aglycones

The objective of this work was to optimise the conditions for the soaking process of soybean for maximal conversion of β-glucoside isoflavones into aglycones using response surface methodology. The investigated variables were the time (3, 4, 6, 8, and 9 h) and temperature (41, 45, 55, 65, and 69 °C ± 1 °C) for the soaking of soybean in 1:1.5 (g:g) ratio with a buffer solution pH 6. The maximum aglycones content (1.22 μmol g⁻¹) in soaked soybeans was estimated and validated at 55 °C ± 1 °C and 6 h of soaking. In this condition, the hydrothermally treated soybean grains showed a minimum content of β-glucoside isoflavones and low β-glucosidase activity. A 30% reduction in total isoflavones content of the system was estimated at 69 °C ± 1 °C not depending on the soaking time, attributed to the thermal instability of malonylglucosides. This work may contribute to the preparation of soy products rich in aglycones from hydrothermally treated soybean.     

Emulsion design for the delivery of β-carotene in complex food systems

β-Carotene has been widely investigated both in the industry and academia, due to its unique bioactive attributes as an antioxidant and pro-vitamin A. Many attempts were made to design delivery systems for β-carotene to improve its dispersant state and chemical stability, and finally to enhance the functionality. Different types of oil-in-water emulsions were proved to be effective delivery systems for lipophilic bioactive ingredients, and intensive studies were performed on β-carotene emulsions in the last decade. Emulsions are thermodynamically unstable, and emulsions with intact structures are preferable in delivering β-carotene during processing and storage. β-Carotene in emulsions with smaller particle size has poor stability, and protein-type emulsifiers and additional antioxidants are effective in protecting β-carotene from degradation. Recent development in the design of protein-polyphenol conjugates has provided a novel approach to improve the stability of β-carotene emulsions. When β-carotene is consumed, its bioaccessibility is highly influenced by the digestion of lipids, and β-carotene in smaller oil droplets containing long-chain fatty acids has a higher bioaccessibility. In order to better deliver β-carotene in complex food products, some novel emulsions with tailor-made structures have been developed, e.g., multilayer emulsions, solid lipid particles, Pickering emulsions. This review summarizes the updated understanding of emulsion-based delivery systems for β-carotene, and how emulsions can be better designed to fulfill the benefits of β-carotene in functional foods.     

Influence of particle size on lipid digestion and β-carotene bioaccessibility in emulsions and nanoemulsions

The interest in incorporating carotenoids, such as β-carotene, into foods and beverages is growing due to their potential health benefits. However, the poor water-solubility and low bioavailability of carotenoids is currently a challenge to their incorporation into many foods. The aim of this work was to study the influence of particle size on lipid digestion and β-carotene bioaccessibility using corn oil-in-water emulsions with different initial droplet diameters: large (d₄₃ ≈ 23 μm); medium (d₄₃ ≈ 0.4 μm); and small (d₄₃ ≈ 0.2 μm). There was a progressive increase in the mean particle size of all the emulsions as they passed through a simulated gastrointestinal tract (GIT) consisting of mouth, stomach, and small intestine phases, which was attributed to droplet coalescence, flocculation, and digestion. The electrical charge on all the lipid particles became highly negative after passage through the GIT due to accumulation of anionic bile salts, phospholipids, and free fatty acids at their surfaces. The rate and extent of lipid digestion increased with decreasing mean droplet diameter (small ≈ medium ? large), which was attributed to the increase in lipid surface area exposed to pancreatic lipase with decreasing droplet size. There was also an appreciable increase in β-carotene bioaccessibility with decreasing droplet diameter (small > medium > large). These results provide useful information for designing emulsion-based delivery systems for carotenoids for food and pharmaceutical uses.     

Rapid method for determination of β-carotene in foods using ultra high performance liquid chromatography

The ultra high performance liquid chromatography (u-HPLC) method for determination of β-carotene in foods was validated in terms of precision, accuracy, and linearity. The u-HPLC separation was performed on a reversed column C₁₈ (particle size 2 μm, i.d. 2 mm, length 50 mm), followed by ultra violet (UV) detection at 450 nm. The recovery of β-carotene was more than 84.4% and the limit of detection and limit of quantitation of u-HPLC analysis were 0.28 and 0.85 μg/mL for β-carotene with butylated hydroxytoluene (BHT) and 0.62 and 1.89 μg/mL for β-carotene without BHT, respectively. The calibration graph for β-carotene was linear from 0.1 to 25.0 μg/mL for u-HPLC. The intra- and interday precisions (relative standard deviations) were <7.5 and <7.8%, respectively. Benefits of u-HPLC analysis of β-carotene in foods is reduction of the analysis time to approximately 1/4, saving the volume of solvent to approximately 1/15. It seems that u-HPLC can offer significant improvements in speed, sensitivity, and resolution compared with conventional HPLC, this bodes well for future applications.