MATERIALS AND METHODS

Materials: Curcumin (CCM) was purchased from Merck Schuchardt (8011 Hohenbrunn bei Munchen). The product had a purity of 94% w/w according to the vendor. Sodium caseinate (NaCas) was from Acros (New Jersey, USA). Other chemicals were obtained from either Sigma-Aldrich or Thermo Fisher Scientific (Pittsburgh, PA). Pasteurized caw whole milk (Juhina^®, Egypt) and skim milk powder Low heat produced by Dairy America, Inc. California, USA. were used for the preparation of the yoghurt, as well as lyophilized microorganism Choozit^® (Danisco, Mexico) containing: Lactobacillus delbrueckii spp. bulgaricus Streptococcus salivarius spp. thermophilus. All employed chemicals exhibited Analar or equivalent quality.

The study was carried out at the Department of Dairy, National Research Centre and Department of Dairy Science and Technology, Menoufia University Shibin El Kom, Egypt from August-October, 2020).

METHODS

Encapsulation efficiency of curcumin: Five milligram of freeze-dried powder was suspended in 10 mL chloroform and was stirred overnight at room temperature (21°C). After centrifugation at 6,000×g for 10 min (Minispin plus, Eppendorf, Hamburg, Germany), the supernatant was transferred and filtered through a PTFE syringe filter with 0.45 μm pore size (Fisher Scientific, Pittsburgh, PA). The permeate was diluted 20 times in chloroform and the absorbance at 419 nm was measured using a UV-Vis spectrophotometer (Evolution 201, Thermo Scientific, Waltham, MA) to determine curcumin concentration based on a calibration curve previously established using standard solutions with different amounts of free curcumin dissolved in chloroform²⁵:

Particle size analysis and microstructure by transmission electron microscopy: The size determination and Particle Dispersity Index (PDI) were recorded by Dynamic Light Scattering (DLS) as described by Soliman and Hassan²⁶. The samples were examined by TEM using a JEOL JEM-1400 Plus TEM with an accelerating voltage of 100 kV at a magnification of 200,000x²⁶. Antioxidant activity determined by The DPPH method was described by Rai et al.²⁷.

Preparation of yoghurt: For the yoghurt preparation, milk was standardized adding 3 g of milk powder per 100 mL of pasteurized milk. Then, a heat treatment was applied by raising the temperature of the milk to 90°C for 20 min and then cooling down to 40-45°C. After cooling at 42°C, the milk was added with the lyophilized culture directly and stirred for 10 min and poured into 100 mL plastic containers, is the same procedure for all samples; then the CCM (both, nanoparticles and free CCM) were added at a 250 mg CCM and stirred for 20 min (120 agitations/min) until complete dissolution. Subsequently, the milk with CCM was incubated at 45°C for 4 hrs until a pH of 4.6 was reached, as well as the control²⁸ yoghurt samples were stored for 14 days at 5±1°C.

Physicochemical analysis: The pH was measured by a digital potentiometer (Beckman, Denver, CO, USA), previously calibrated, at room temperature. Moisture content was determined through water evaporation method 16.032, AOAC²⁹. Acidity was quantified by titration of 9 mL of the sample using phenolphthalein and NaOH (0.1 mol Equi L^–1) method 16.023, AOAC²⁹.

The Texture Profile Analysis (TPA) was determined using a Texture Analyzer (Mult- test 1d Memesin, Food Technology Corporation, Slinfold, W. Sussex, UK) Measuring the double compression force (g) in all samples of yoghurt (100 mL, mm height) using a cylindrical body of 4.3 cm in diameter, descending at a speed of 1 mm sec^–1 and reaching a depth of 20 mm. All measurements were carried out at a temperature of 20°C by triplicate³⁰.

Evaluation of sensory properties: The yoghurt samples were organoleptically evaluated as described by Hamed et al.³¹. Some panellists from the staff members of the Department of Dairy Science, National Research Center, Egypt, were evaluated each yoghurt sample and used a quality rating scorecard for evaluation of flavour (60 points) and body and texture (30 points) and appearance (10 points) as described by Hamed et al.³¹.

Statistical analysis: The analyses of prepared samples were conducted at least in triplicates; comparisons of the treatments were completed by one-way ANOVA and Tukey’s tests by SPSS, ver. 16.0 statistics programs. A 95% minimum confidence level was taken for all statistical analyses³².

RESULTS AND DISCUSSION

Particle size, polydispersity index and encapsulation efficiency of CSCCMNPs: The result of Table 1 showed that the particle size of dispersions prepared from NaCas nanoparticles and CSCMMNPs containing 0.25, 0.50 and 1.00 g CMM. The particle size of the NaCas nanoparticles 83.27 nm and the size of the dispersions CSCCMNPs with curcumin additions (98.10, 125.70, 184.20 nm) respectively were significantly larger than that of the NaCas nanoparticles. Particle polydispersity of Casein nanoparticle of 0.213, increased to 0.480 by an increase of addition CCM to 1 g/200 mL. de Kruif and Huppertz³³ found bovine casein micelles to be very monodisperse and are a Calculated polydispersity of 0.213-0.480. Encapsulation Efficiency of CCM of (0.25, 0.50 and 1.00 g/200 mL NaCS) CMM was encapsulated by Caseinate of 97.00, 91.25 and 82.90% illustrated in Table 1. This increased with an increase in the CCM ratio. Heat treatment of casein in ethanol 40% at 60°C has changed casein structurally by polarity that affects solvent quality and thus dissociation and re-association of caseins³⁴.

Fig. 1:

Transmission electron micrograph of CSCCMNps

Fig. 2:

Trolox Equivalent Antioxidant Capacity (TEAC) of the NaCas nanoparticles, free CCM and encapsulated CCM ratio (0.25, 0.50 and 1 g CCM) CSCCMNPs at 30°C

This was consistent with our monitoring of decrease turbidity. The heat treatment of casein in ethanol at 40% increases the availability of hydrophobic ligands of casein for binding with CCM through blending. Also, caseins have open structures containing numerous proline residues evenly distributed throughout their amino acids and are very tendentious as binders of bioactive compounds such as phenolic compounds and antioxidants³⁵. The Encapsulation Efficiency (EE) obtained from curcumin in NaCas NPs can be compared with EE of curcumin nanoencapsulation in zein using electrohydrodynamic atomization it’s the range of 85-90%³⁶.

The microstructure of CSCCMNps was examined by TEM as shown in Fig. 1. TEM micrographs revealed that the size of CSCCMNps in the micrograph was not consistent with the results of DLS. We find that the size of the CSCCMNPs is less than the results of DLS because the measurement is done under a vacuum that may lose moisture and thus reduce the size.

Antioxidant activity of CSCCMNPs: Figure 2 showed the TEAC of curcumin before and after encapsulation. The TEAC of the NaCas nanoparticle was also determined, which was 307 μM. Also, the antioxidant activity has been calculated for caseins that are attributing to amino acid remains like tryptophan, cysteine and methionine that can be oxidized by free radicals^37,38. The antioxidant activity of free curcumin had been low (260 μM TEAC) when thawed in ethanol followed by mitigation in distill water for analysis because the limited solubility of curcumin had effected to bind with free radicals. Although, curcumin probably had phenolic –OH groups which play the main part in oxidation reactions³⁷. The antioxidant activity for different ratios from CCM (0.25, 0.50 and 1.00) loaded at NaCS is 553, 713 and 907 μM TEAC respectively. Which, higher than obtained from free CCM and dispersing NaCsNPs. Takahashi et al.³⁹ have been reported that the enhancement of the antioxidant activity of CCM after nanoencapsulation in dispersion colloids such as lecithin and β-casein.

Yoghurt properties: The result of this study has shown significant differences (p<0.05) in physicochemical characteristics which increased total solids and protein, in fortified yoghurt by CSCMMNPs of (15.27 and 4.73%) than control yoghurt is (14.19 and 3.80%) respectively (Table 2). While has shown non-significant (p<0.05) differences between control yoghurt and fortified with free CCM. Because of the addition of CSCMMNPs nearly 1 g protein but the addition of free CCM nearly 250 mg. The results agree with the obtained by Ghorbanzade et al.⁴⁰.

In yoghurt, a decrement of pH indicates the release of lactic acid in the medium by Lactic Acid Bacteria (LAB). The data of Table 3 showed yoghurt samples' evaluation of pH and acidity changes during 14 days of storage at 5°C. The pH values showed a trend to decrease (p<0.05) during the storage period in all samples but, the acidity values increased significantly through 14 days of storage. Where the control pH decreased from 4.65 at fresh to 4.56 after 14 days of cold storage.

But the acidity increased from 0.76-0.82. We found no significant differences (p<0.05) between all treatments for pH and acidity determination. These results also were in agreement with Bonczar et al.⁴¹.

Texture profile analyses simulate the conditions of a product in the mouth by compressing. The results in Table 4 showed that the highest hardness, Springiness, Cohesiveness, Gumminess and Chewiness were measured in yoghurt fortified with CSCCMNPs of 91.77 g, 0.77 mm, 0.61, 55.98 g and 43.10 g*mm respectively. Followed by, yoghurt fortified with free CCM is 81.58 g, 0.73 mm, 0.59, 47.54 g and 34.71 g*mm respectively, while the lowest one was observed in control yoghurt is 79.85 g, 0.68 mm, 0.58, 46.31 g and 31.49 g*mm respectively. The physical properties of yoghurt gels including gel stiffness and permeability, rearrangement of protein particles in gel network and structure breakdown of stirred-type yoghurts are important factors that influencing the physical and structural properties of yoghurts²⁸.

Table 5 shows the sensory properties of control yoghurt and fortification at 5±1°C. There was a significant difference (p>0.05) in the Flavour, body and texture, colour and appearance score. The CSCCMNPs scored highest for all parameters, followed by fortified with free CCM and the lowest-scoring control yoghurt.

Finally, the caseinate gives the ability to bind CCM by desolvation methods and freeze-drying for warm caseinate solutions. Also, the results indicated that fortification of yoghurt with CSCURNPs had no adverse effect on the organoleptic properties of resultant yoghurt. Besides, body, taste and texture were improved and the enhancement of yoghurt acceptability. Foda et al.⁴² noticed that less amount of turmeric in yoghurt given more acceptable and enhancement rheological properties of set yoghurt.

Caseinate gives the ability to bind CCM hence gives the ability to apply in dairy and pharmaceuticals products. CSCURNPs give more acceptable and enhanced rheological properties and texture of yoghurt.

CONCLUSION

Caseinate curcumin nanoparticles were prepared using a novel technique by freeze-drying for warm caseinate solutions. This improves the ability to load and bind curcumin and enhances the solubility of it. The results indicated that fortification of yoghurt with CSCURNPs had an enhancement to the organoleptic properties of resultant yoghurt. Besides, body and texture were improved and the enhancement of yoghurt's sensory acceptability.

SIGNIFICANCE STATEMENT

The present study found the heat treatment of casein in 40% ethanol increases the availability of hydrophobic ligands of casein for binding with CCM through blending. Also, caseins have open structures containing numerous proline residues evenly distributed throughout their amino acids and are very tendentious as binders of hydrophobic components as curcumin. Also, can applicable CSCCMNps in prepared yoghurt, because of her therapeutic properties. Furthermore, CSCCMNps improved texture profile analysis, antioxidant activity and sensory acceptability and technological properties of the functional yoghurt. Subsequently, we recommended using CSCCMNps in functional dairy products.