Maillard-reaction based nano-capsules for protection of water-insoluble nutraceuticals in clear drinks.

Transcription

1 Maillard-reaction based nano-capsules for protection of water-insoluble nutraceuticals in clear drinks. Gilad Markman, Yoav D. Livney The Faculty of Biotechnology and Food Engineering, The Technion, Israel Institute of Technology, Haifa 32000, Israel ABSTRACT One of the most important aims of contemporary food engineering is the enrichment of foods with health enhancing components. There is a growing public awareness for healthy nourishment that should include necessary daily amounts of required micronutrients. Moreover, there is demand for natural ingredients and processes. Integrating a hydrophobic nutraceutical (HN) in aqueous media, particularly clear beverages, is a challenging task. The complications associated with such systems are poor HN solubility and stability, which are usually improved by addition of emulsifiers. One of the emulsifiers traditionally utilized is gum arabic which is a natural protein-polysaccharide conjugate (PPC). Its main disadvantages are its high price and highly variable composition, quality and availability. Therefore, many efforts are aimed at finding good inexpensive substitutes. Producing PPC may be done by enzymatic, chemical or electrosynthesis reactions. A particularly attractive way to form PPC is via Maillard reaction, achieved only by heating, typical of cooking and food processing. We formed casein (CN)-maltodextrin (MD) Maillard reaction conjugates and used them to nanoencapsulate hydrophobic and hydrophilic nutraceuticals. Clear solutions were obtained with nanoencapsulated VD. Particle size of VD loaded PPC nanoparticles was much smaller than that of MD-caseinate mixture with VD; e.g. at 3: VD:CN molar ratio, 7 of the mixture-based nanoparticles were >00 nm, while ~0 of the PPC-based nanoparticles at this ratio were <00 nm. Unencapsulated VD at same concentrations was very turbid. Conjugation significantly improved the protection against oxidation and low ph conferred to VD, important for acid drinks, and for survival through gastric digestion. PPC protection conferred to epigallocatechin-3-gallate (EGCG) was also demonstrated. Overall the study showed the very good potential of Maillard conjugates of proteins and oligosaccharides for nanoencapsulation of nutraceuticals for clear drinks application, good solubilization, stabilization and protection conferred to the sensitive bioactive compound against degradation during shelf life, and gastric digestion. Keywords: nanoencapsulation; Maillard reaction; protein-polysaccharide conjugates; vitamin D; Epigallocatechin-3- gallate. INTRODUCTION In recent years there has been growing public awareness for the importance of a healthy diet. A promising approach for addressing this need is the enrichment of widely consumed foods and beverages with nutraceuticals [-2]. Many of the bioactives desired for enrichment are hydrophobic, but the enrichment of clear beverages with hydrophobic nutraceuticals (HN) is challenging for several reasons, mainly their poor water-solubility, as the particles formed must be small enough and colloidally stable to maintain clarity of the beverage. Moreover, oxidation sensitive nutraceuticals (both hydrophobic and hydrophilic) must be protected against degradation during product processing, shelf-life and digestion. Protein-Polysaccharide conjugates covalently bonded by the Maillard reaction have been shown to be emulsifiers with better surface activity and emulsion stabilization capacity than the proteins used to form them [3-4], but their use as nanoencapsulators in clear systems has apparently not yet been reported. The main goal of this study was to develop protective nanovehicles based on Maillard reaction based proteinpolysaccharide conjugates (PPC), for the enrichment of clear beverages with sensitive nutraceuticals. We used caseinate, an amphiphilic protein, and maltodextrin to form PPC. Our specific objectives were:. To find a preferred method for formation of Maillard reaction conjugates. 2. To load the nanovehicles with model nutraceuticals, e.g. the hydrophobic Vitamin D (VD) and the hydrophilic Epigallocatechin-3-gallate (EGCG), and evaluate particle size distribution. 3. To evaluate the protection conferred by the nanovehicles to the model nutraceuticals 4. To evaluate the release of nutraceuticals during simulated gastric digestion.

2 MATERIALS & METHODS PPC formation: Freeze dried solutions of sodium caseinate and MD at different molar ratios were heated (60 o C at 79% RH) for 4, 6, and 8 hrs. Conjugation was confirmed and quantified using SDS PAGE and OPA (Ortho-phthaldialdehyde) assay. Conjugate separation was done by acidifying the solution with HCl to ph=4.6 (the isoelectric ph of CN), followed by centrifugation at 000g for 0 minutes. The ph was adjusted to 7.0 with 5N NaOH and conjugates were freeze-dried. Conjugates were dissolved in buffers of ph 2.5, 4.6 or 7.0. VD solution in ethanol was added to a PPC solutions while vortexing (final ethanol concentration was 0.25%v/v). Size distribution of the particles formed was measured using dynamic light scattering (DLS). Protection of conjugates against VD degradation during simulated shelf life at 4 C and ph 7 was evaluated. Residual VD concentration was measured by extraction and quantification by RP-HPLC. Protection against EGCG degradation during shelf life at ph=7 and room temperature was studied on samples of initial EGCG concentration of 0.9 mg/ml. EGCG degradation was measured spectrophotometrically at 425nm as previously described [5]. NR release during simulated gastric digestion was evaluated by adding it to buffer, mixture and conjugate solutions (3mg/ml CN) at ph=2.5, then adding pepsin to some of the solutions (:20 pepsin: CN mass ratio). The solutions were stirred for 2 hrs in a 37 o C water bath. Released NR was adsorbed to the glass and quantified by emptying the vials, and dissolving the adsorbed NR by adding water: acetone (:) to the empty vials, and measuring NR fluorescence in the acetone solution. RESULTS & DISCUSSION A confirmation of the successful conjugation was achieved by both OPA and SDS-PAGE assays. OPA results showed 55% decrease in the number of free amine residues, suggesting more than half the available amines participated in the Maillard reaction. Figure presents SDS-PAGE results: casein-only was in lane 7, while samples in lanes 5-6 were mixtures of casein and MD, heated separately, then mixed. After conjugation (lanes -4), the whole casein band shifted upward to larger Mw, providing an excellent evidence for the formation of Maillard conjugates. The shift of about 5-0kDa compared to the casein band (lane 7) suggests of an average conjugation of ~ MD molecule per casein had occurred, although weak bands of higher Mw appeared too, possibly due to conjugation of several MD to one casein molecule. Figure. SDS PAGE. Lanes: - 4 conjugates: - MD:CN=8, 2- MD:CN=4, 3- MD:CN=2, 4- MD:CN=; lanes 5-6- mixture: 5- MD:CN=8, 6- MD:CN=, 7- CN, 8-size marker. Conjugation time=8 hrs Isoelectric precipitation- The yield of isoelectric separation was defined as follows: supernatant absorbance (278nm) Yield = 00 pre separated conjugate solution absorbance (278nm).

3 In Figure 2 it is seen that more than of the conjugates which formed during 8 hours of heating did not sediment at ph=4.6 (isoelectric ph of Casein). The yield of the 6 hr heating conjugates was smaller (3-7%) but it was still better than the yield of the mixture, which was only 5-2%, due to the precipitation of casein at its pi. 3 con 8 hrs mix 8 hrs con 6 hrs mix 6 hrs % Yield MD: CN molar ratio Figure 2. Separation Yield at ph=4.6 Next, we evaluated the nanoencapsulation of HN, in terms of transparency and particle size distribution obtained. Freeze-dried supernatant conjugates (4: MD:CN, 8hr heating) were dissolved in different buffer solutions (ph 2.5, 4.6, and 7.0), and VD, dissolved in ethanol, was added while stirring. Size distributions of the particles formed at these ph values are shown in Figure 3. ph=2.5 ph=4.6 ph=7 0,8 % Volume 0,6 0,4 0, Diameter (nm) Figure 3. Size distribution of VD-loaded CN-MD conjugates, following isoelectric separation, freeze-drying and dissolution at different ph values. Casein concentration=mg/ml; CN:VD molar ratio =:. As can be seen in Figure 3, at ph of 2.5, 4.6 and 7, conjugate-vd particle diameter was less than 30 nm and solutions were completely clear. VD added at the same concentration to the buffers yielded very turbid dispersions.

4 Particle size distribution of un-separated conjugates- We also evaluated the particle size distribution of VDloaded conjugates which were not separated at ph 4.6, as the additional steps, and low yield of this purification process may not be justified for some applications. Figures 4 and 5 show that particle size of VD loaded PPC nanoparticles was much smaller than the particle size of a control MD-caseinate mixture with VD. E.g. at 3: VD:CN molar ratio, 7 of the mixture-based nanoparticles were larger than 00 nm, while ~0 of the PPC-based nanoparticles at this ratio were smaller than 00 nm. Volume-weighted percent diameter<00 nm diameter>00 nm average diameter Vitamin D : casein molar ratio Figure 4. Size distribution of conjugate + VD3 at different VD:CN molar ratios. Casein concentration was mg/ml and the ph was 7. The left y axis refers to the bars, and right y axis refers to the circles Average diameter(nm) diameter<00 nm diameter>00 nm average diameter Volume-weighted percent Vitamin D : Casein molar ratio 00 0 Average diameter (nm) Figure 5. Size distribution of mixture + VD3 at different VD:CN molar ratios. Casein concentration was mg/ml and the ph was 7. The left y axis refers to the bars, and right y axis refers to the circles. The range of ratios described in Figure 5 was only up to VD: CN = 5, because at higher VD concentrations the solution was too turbid and could not be analyzed by the DLS.

5 Protection against degradation- As can be seen in Figure 6 the conjugates conferred much better protection to VD against degradation during simulated product shelf life than the mixture or the buffer. 0 Residual VD 65% 3 con mix buffer Time (days) Figure 6. Residual VD with time, in conjugate, mixture of MD and casein or buffer. Casein concentration was 3 mg/ml; initial VD concentration was 0.05 mg/ml; MD: Casein molar ratio=4; VD: Casein molar ratio=; ph=7. Trendlines are drawn as a first order approximation. EGCG was also better protected by the conjugates compared to the controls as can be seen in Figure 7. cas mix buffer 7 MD con ΔO.D 425nm (EGCG oxidation) 0,8 0,6 0,4 0, Time (hrs) Figure 7. EGCG degradation as a function of time at room temperature, with and without the different protective systems studied. All solutions were at ph=7; Initial EGCG concentration was 0.9 mg/ml; casein concentration was 5 mg/ml.

6 Release of NR during simulated gastric digestion- Based on the phenomenon that when added to pure water, NR rapidly adsorbs to the glass vial[6], we quantified the release of NR from the nanocapsules, as the amount adsorbed to the glass. This enabled both the quantification of the encapsulation efficiency in this case, and the release during simulated digestion. As can be seen in figure 8, the encapsulation efficiency in the conjugates was about 92% and in the mixture about 9. Interestingly, NR was not released from the conjugates during simulated gastric digestion, suggesting potential as enteric coating. % Nile red adsorbed to glass Buffer Mix Con Buffer Mix Con No pepsin With pepsin Figure 8. Percent of NR adsorbed to glass in con/mix/buffer solutions, with and without pepsin after 2hrs at 37 o C; ph=2.5. CONCLUSIONS We have successfully demonstrated the protection of hydrophobic and hydrophilic nutraceuticals using Maillard reaction based CN-MD conjugates which can potentially be used to enrich clear beverages. Conjugates conferred better protection than casein-md mixture against vitamin D and EGCG oxidation. PPC protection to HN at gastric conditions was found, suggesting potential as enteric coating. REFERENCES [] Sagalowicz L. & Leser M. E Delivery systems for liquid food products. Current Opinion in Colloid & Interface Science, 5(-2), [2] Zimet P. & Livney Y. D Beta-lactoglobulin and its nanocomplexes with pectin as vehicles for omega-3 polyunsaturated fatty acids. Food Hydrocolloids, 23(4), [3] Oliver C. M., Melton L. D. & Stanley R. A Creating proteins with novel functionality via the Maillard reaction: A review. Critical Reviews in Food Science and Nutrition, 46(4), [4] Dickinson E Hydrocolloids as emulsifiers and emulsion stabilizers. Food Hydrocolloids, 23(6), [5] Shpigelman A., Israeli G. & Livney Y. D Thermally-induced protein-polyphenol co-assemblies: beta lactoglobulin-based nanocomplexes as protective nanovehicles for EGCG. Food Hydrocolloids, 24(8), [6] Sackett D. L. & Wolff J Nile red as a polarity-sensitive fluorescent probe of hydrophobic protein surfaces. Analytical Biochemistry, 67(2),