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Keywords:

  • Algae oil;
  • bovine casein;
  • caprine casein;
  • microbial transglutaminase;
  • microencapsulation;
  • n-3 fatty acids;
  • oxidative stability

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgments
  8. References

Bovine and caprine caseins were cross-linked with microbial transglutaminase (mTG). The mTG-cross-linked bovine or caprine casein dispersion, mixed with 14.5% maltodextrin (DE = 40), was used to prepare emulsions with 10.5% algae oil. Oxidative stability of emulsions was evaluated by peroxide values (PVs) and anisidine values. Adding liposoluble rosemary extract rich in carnosic acid and δ-tocopherol lowered the formation of hydroperoxides and their subsequent decomposition products in emulsions. Emulsions stabilised with liposoluble rosemary extract rich in carnosic acid and δ-tocopherol were spray-dried at 180/95 °C. Algae oil microencapsulated with mTG-cross-linked bovine casein reduced PV by ≈ 34%, while the algae oil microencapsulated with mTG-cross-linked caprine casein with low levels of αs1-casein reduced PV by ≈ 42% at 4 weeks of storage at 30 °C. The investigation suggests that liposoluble rosemary extract rich in carnosic acid and δ-tocopherol effectively protected algae oil during the coating process with mTG-cross-linked bovine and caprine caseins. The above results clearly indicated that the choice of milk caseins (bovine vs. caprine) cross-linked with mTG impacts the oxidative stability of spray-dried algae oil emulsions (microcapsules) enriched with n-3 fatty acids.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgments
  8. References

Long-chain n-3 fatty acids docosahexaenoic acid (DHA, C22:6) and eicosapentaenoic acid (EPA, C20:5) are essential lipid components of cellular membranes in all tissues (Valentine & Valentine, 2004; Kidd, 2007). Both fatty acids are essential for growth and development, and DHA is particularly important for normal visual and cerebral function (Kidd, 2007; Gogus & Smith, 2010). Typical dietary sources of long-chain n-3 fatty acids include fish, shellfish and several microalgae. Rich sources of the short-chain n-3 fatty acid α-linolenic acid (ALA, C18:3) include walnuts, standard soy and canola oils and flaxseed. ALA is converted through a series of desaturation and elongation steps to DHA and EPA. Elongation and desaturation of ALA to DHA and EPA occurs in human leucocytes and in the liver of both humans and rodents (e.g. rats). The n-3 and n-6 fatty acids compete for the desaturation enzymes. But both the δ-4 and δ-6 desaturases (the enzymes involved in desaturation) prefer the n-3 to the n-6 fatty acids (Emken et al., 1999).

Generally, the North American and Western European diets are deficient in n-3 fatty acids and comparatively have an enormous amount of n-6 fatty acids due to changes in feeding practices of livestock and the development of techniques that increase the production of vegetable oils rich in n-6 fatty acids (Lands et al., 1990). Therefore, a need exists for n-3-fortified food products. However, n-3 fatty acids are more prone to oxidation (Harris, 2007). Typical initiators of oxidation are air, increased temperatures, contact with transition metals and exposure to light. Oxidation not only produces rancid flavours but can also decrease the nutritional quality and safety of n-3-fortified foods, which may play a role in the development of diseases. Use of new ingredients and technologies can minimise the negative effects of lipid oxidation. The most promising technique is microencapsulation, a process that surrounds small droplets of oil with a protective matrix, usually made from proteins and/or carbohydrates (Gouin, 2004). This not only protects the oil from oxygen exposure, but depending on the encapsulating material and its preparation techniques can protect the taste buds from the oily taste. To improve stability of n-3 fatty acids, antioxidants can be added to either the oil or the shell material (Jacobsen et al., 2008).

The bovine caseins (αs1-, αs2 -, β- and κ-casein) have applications in a spectrum of food products including baked products and cereals, desserts, coffee creamers and pasta products (Southward, 1989). Caseins possess various emulsifying, water-binding and whipping/foaming properties. The different casein components have a strong tendency to self-association and complex formation, which reflects their tendency to form micelles in milk (Farrell et al., 2004).

Bovine and caprine caseins have been found to stabilise n-3 fatty acids in algae oil-in-water emulsions against oxidation (Mora-Gutierrez et al., 2010). This was attributed to their chelating and free radical scavenging properties as well as their ability to form a thick layer at the interface. A recent study by Bao et al. (2011) shows that oxidative stability of microcapsules formed by spray-drying of algae oil can be improved with the cross-linking of the bovine casein layer using microbial transglutaminase (mTG) over 60-min incubation at 45 °C. The enzyme mTG catalyses formation of intramolecular or intermolecular isopeptide linkage between glutamyl and lysyl residues within the target proteins (Christensen et al., 1996). Considering the variation in casein composition from caprine sources, the mTG cross-linking of caprine caseins may offer a better encapsulating material for n-3 fatty acids (Mora-Gutierrez et al., 1991). In this study, we evaluated the oxidative stability of algae oil microencapsulated by mTG-induced cross-linked caprine caseins with high and low levels of αs1-casein. It was also investigated whether the protective antioxidant rosemary extract rich in carnosic acid could be observed in algae oil microencapsulated in cross-linked caprine caseins induced by mTG. Bovine casein was used for comparison purposes.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgments
  8. References

Reagents, chemicals and natural products

Algae oil, a rich source of n-3 fatty acids composed of 3% EPA and 46% DHA, was obtained from Martek Biosciences, Inc. (Boulder, CO, USA). Liposoluble rosemary extract containing a minimum from 15 to 30% carnosic acid and a maximum of 10% carnesol was obtained from Biolandes Arômes (Boulogne, France). Maltodextrin (DE = 40) was obtained from Grain Processing Corporation (Muscatine, IA, USA). Microbial transglutaminase was obtained from Ajinomoto (Paramus, NJ, USA). Soya bean lecithin was a gift from ADM (Decatur, IL, USA). Carnosic acid and δ-tocopherol were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All reagents used were of analytical grade, ACS certified or HPLC grade from Fisher Scientific (Pittsburg, PA, USA). Deionised water was used in all experiments.

Sample preparation and analysis

Purification and stabilisation of the algae oil

Algae oil was purified chromatographically to remove tocopherols, other antioxidants and carotenoids, as previously described (Lampi & Kamal-Eldin, 1998). The chromatographically purified algae oil was not significantly changed in fatty acid composition containing a DHA concentration of 42.9%. The chromatographically purified algae oil was void of tocopherols, carotenoids and peroxides.

The chromatographically purified algae oil with and without the liposoluble rosemary extract rich in carnosic acid (1250 mg kg−1 oil) was stabilised by the addition of δ-tocopherol (1000 mg kg−1 oil) previously dissolved in methanol. Methanol was removed under a stream of nitrogen before the addition of the chromatographically purified algae oil.

Preparation of bovine and caprine caseins

Caseins were obtained from the milk of a Jersey cow and French-Alpine goats. The samples of caprine milk were collected from individual French-Alpine animals, which were raised at the International Goat Research Center in Prairie View A&M University, TX, USA. The caprine milk caseins were selected based on yielding high and low levels of αs1-casein as determined by reversed-phase high-performance liquid chromatography (RP-HPLC) (Mora-Gutierrez et al., 1991). Caseins were isolated from 2 L of fresh, uncooled milk to which phenylmethanesulphonyl fluoride (0.1 g L−1) was added immediately to retard proteolysis. The milk was centrifuged at 4000 g for 10 min at room temperature to remove the cream fraction. Skim milk (500 mL) was diluted with an equal volume of distilled water and warmed to 37 °C. Casein was precipitated by gradual addition of 1 N HCl to pH 4.5. The precipitate was homogenised with a hand-held homogeniser (Biospec Products Inc., Bartlesville, OK, USA) at low speed and dissolved by the addition of 1 m KOH to yield a solution of pH 7.0. The casein was reprecipitated, washed and then resuspended. The casein was subsequently cooled to 4 °C and centrifuged at 100 000 g for 30 min to remove residual fat. The integrity of the samples was confirmed by SDS-PAGE, and densitometry was used to assess the relative concentration of casein components (Basch et al., 1989).

In addition, the bovine and caprine casein samples were analysed to determine protein (N × 6.38), moisture, lactose and ash contents (AOAC, 2005). The amino acid content of the bovine and caprine casein samples was measured at The Protein Technologies Laboratory, Department of Entomology, Texas A&M University (College Station, TX, USA). The results were all corrected to a moisture- and ash-free basis. All samples were analysed in duplicate.

Emulsion preparation

The casein (5% w/v) was dispersed in deionised water and then incubated with mTG (4U g−1 casein) for 60 min at 45 °C with constant shaking. Immediately after treatment, mTG was inactivated by freezing the mixture for 10 min. The non-mTG-incubated samples (0 min) were considered as control. Maltodextrin (14.5% w/v) and algae oil (10.5% w/v) were added to cross-linked casein solutions. Oil-in-water emulsions were made by blending the lipid and aqueous phase for 3 min using a hand-held homogeniser. The coarse emulsion was then homogenised three times at 40, 60 and 80 MPa and at a temperature of 50 °C through a high-pressure TC5 homogeniser (Stansted Fluid Power, Harlow, UK).

Determination of mean droplet size

The particle size distribution (d3,2; μm) of the oil droplets (μm) in the algae oil-in-water emulsions (with and without mTG) was determined by laser diffraction with a SALD-2101 laser diffraction particle analyser (Shimadzu, Columbia, MD, USA) after homogenisation at 0 and 48 h. The mean of three replicates was reported.

Emulsifying efficiency

To assess emulsifying efficiency of the bovine and caprine caseins in algae oil-in-water emulsions, we have used two procedures: emulsifying activity index (EAI) and emulsifying stability index (ESI). The EAI and ESI were determined according to turbidometric techniques, as previously described (Pearce & Kinsella, 1978; Chove et al., 2001).

Freshly prepared algae oil-in-water emulsion (100 μL) was diluted in 9.9 mL of deionised water (100-fold dilution). Then 800 μL of the diluted emulsion was added to 3.2 mL of 0.1% SDS, resulting in a final 500-fold dilution. The absorbance of the final dilutions was measured at a wavelength of 500 nm at 0 (A0) and 10 (A10) min in a 1-cm path length cuvette with a UV/vis model DU-530 spectrophotometer (Beckman Instruments Inc., Fullerton, CA, USA). The EAI was calculated as follows:

  • display math

where = 2.303, A0 is the absorbance of the diluted emulsion immediately after homogenisation, the dilution factor is 500, Φ is the volumetric fraction of oil, 0.25 in this case, C is the weight of protein per unit volume (g mL−1) in the aqueous phase before the emulsions were formed and 10 000 is the correction factor for square metres. The EAI of emulsion was monitored for 0 and 48 h. The mean of three replicates was reported.

The ESI of algae oil-in-water emulsions was calculated as follows:

  • display math

where ∆A is the change in absorbance between 0 and 10 min (A0A10) and t is the time interval, 10 min. The mean of three replicates was reported.

Fluorescence studies of protein–phenolic compound interaction

Fluorescence studies of protein–phenolic compound interaction were carried out after dissolving 500 μL of emulsion in citrate buffer (1 mL). For this purpose, oil-in-water emulsions containing 1% lecithin and 10% algae oil stripped of δ-tocopherol and liposoluble rosemary extract rich in carnosic acid were prepared in 25 mm citrate buffer (pH 6.7) as described above. Carnosic acid was added to methanol solution in screw-capped 50-mL Erlenmeyer flasks and then methanol was removed under a stream of nitrogen before the addition of the oil-in-water emulsion (5 mL). The control and mTG-modified caseins were then added. Samples were subsequently sonicated for a total dispersion of the caseins and phenolics for 5 min. The fluorescence studies were performed on a Shimadzu spectrofluorometer RF-5000 (Shimadzu Corporation, Kyoto, Japan) equipped with a calculator and plotter. The casein was excited at 280 nm, and the emission spectra were recorded between 300 and 400 nm. Emission spectra were recorded in the concentration range mentioned in the legend of Fig. 3.

Microencapsulation by spray-drying

The casein-based, algae oil-in-water emulsions stabilised with δ-tocopherol and liposoluble rosemary extract rich in carnosic acid were spray-dried in a laboratory-scale spray drier (Armfield Ltd., Hampshire, UK) equipped with 0.5-mm-diameter nozzle atomiser. The inlet and outlet temperatures were maintained at 180 and 95 °C, respectively. The microcapsules so prepared were collected from the collecting chamber, filled in loosely capped brown glass containers and stored at 30 °C and 33% relative humidity for 0, 1, 2, 3 and 4 weeks. After the desired storage period, glass containers were frozen at −30 °C until determination of peroxide value (PV).

Microencapsulation efficiency

To assess the efficiency of entrapment of DHA within the casein matrix, surface DHA oil was extracted from the spray-dried powders (3 g) by adding 30 mL of petroleum ether followed by mixing with a vortex for 1 min at 21 °C. The mixture was filtered through Whatman No. 2 filter paper into a preweighted 100-mL round-bottom flask. The solvent was evaporated using a rotary evaporator. The DHA oil was extracted by dispersing 10 g of spray-dried powders in deionised water and extracting the DHA oil with hexane/isopropanol (90:30, v/v) mixture. The bottles were shaken for 15 min at 160 rpm using an automatic shaker and centrifuged for 5 min. The clear organic phase was collected, and the aqueous phase was re-extracted with the solvent mixture. The collected clear organic phase was filtered through anhydrous sodium sulphate into a 250-mL round-bottom flask. The solvent was evaporated using a rotary evaporator, and the lipid content was determined gravimetrically (Klinkesorn et al., 2006). Microencapsulation efficiency (ME) was calculated as follows:

  • display math
Measurement of PV

Peroxide value was determined as a measure of lipid oxidation in the liquid emulsions and microcapsules. Lipids were extracted from the microcapsules according to the method of the American Oil Chemist's Society (AOCS) Official method Cd 8-53 (AOCS, 2009) as follows: 10 g of microcapsules was weighed in a 250-mL Erlenmeyer flask, and 20 mL of chloroform was added followed by 30 mL of glacial acetic acid. The content was stirred to dissolve the algae oil. Saturated KI solution (0.5 mL) was added, and the flask was left to stand for 1 min and then gently mixed. Deionised water (30 mL) was added, and the content was titrated against 0.01 N Na2S2O3 using 0.5 mL 1% starch indicator under constant magnetic stirring. Blank analyses were performed in the same way without addition of microcapsules. PV was calculated as follows:

  • display math

where S is sample titre (mL); B is blank titre (mL); and N is the normality of Na2S2O3 The mean of three replicates was reported.

Measurement of anisidine value

Anisidine value (AV) was determined in the liquid emulsions and microcapsules according to AOCS Official Method Cd 18-90 (AOCS, 1998). This method determines the amount of aldehydes (principally 2-alkenals and 2,4-dienals) present in the oil. The mean of three replicates was reported.

Protein oxidation

Casein oxidation in the algae oil-in-water emulsions was measured by fluorescence spectroscopy (Heinonen et al., 1998). Emission spectra of protein oxidation (carbonyls) were recorded from 400 to 500 nm with the excitation wavelength set at 350 nm.

Statistical analysis

In each experiment, the results of triplicate analyses were used to test experimental variables. The data were analysed by anova using PRO GLM of SAS (version 8.2; SAS Institute, Cary, NC, USA). The least significant test of SAS was used to determine significant differences between means at P < 0.05.

Results and discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgments
  8. References

Effect of type of casein on emulsion stability

One factor contributing to the differences in antioxidant activity of the bovine and caprine caseins in algae oil-in-water emulsions is the different profile of caseins (Mora-Gutierrez et al., 2010). Bovine and caprine caseins both contain the four main casein classes: αs1-, αs2 -, β- and κ-casein, but the level of αs1-casein in caprine caseins isolated from the milks of American-bred, French-Alpine goats ranges from high to low (Mora-Gutierrez et al., 1991). The bovine and caprine caseins used in this study had different compositions in terms of αs1-, αs2 -, β- and κ-casein (Table 1). Because of the compositional differences, these milk proteins exhibit major differences (P < 0.05) in terms of the content of potential antioxidative amino acids histidine, methionine, phenylalanine, proline, tryptophan and tyrosine (Fig. 1); curiously, these differences favour the bovine casein, which in previous studies was less effective in suppressing lipid oxidation (Mora-Gutierrez et al., 2010). It was expected that a high content of antioxidative amino acids in the caseins would lead to a decrease in lipid oxidation. Instead, a high content of the β-casein fraction was correlated with improved oxidative stability of algae oil-in-water emulsions (Mora-Gutierrez et al., 2010). The results by Mora-Gutierrez et al. (2010) were largely explained on the basis of interfacial phenomena having a significant effect on oxidative stability of lipid systems. The mechanism of this process may be related to the affinity of β-casein towards the oil-in-water interfaces in emulsions, that is, β-casein was adsorbed and formed a viscoelastic film at the oil-in-water interface of the emulsion. As caprine casein has a much higher content of the β-casein fraction than bovine casein (Table 1), it would be strongly absorbed and form a film at the interface. Thus, in the algae oil-in-water emulsions, the caprine caseins high and low in αs1-casein were more protective against oxidation (Mora-Gutierrez et al., 2010). In addition, there were no significant differences (P < 0.05) in the amino acids targeted by mTG: glutamate (the sum of the target glutamine and glutamate) and lysine (Fig. 1). These bovine and caprine caseins had similar compositions in terms of protein, moisture, lactose and ash contents (Table 1). To evaluate alternative sources of milk proteins (bovine caseins) cross-linked with mTG, the caprine caseins with high and low levels of αs1-casein (Table 1) were compared in the algae oil-in-water emulsion system.

Table 1. Composition of bovine and caprine caseins
 BovineCaprine
High in αs1-caseinLow in αs1-casein
  1. Values represent the mean ± SD of duplicate measurements. Means in the same row with different letters a–c are different (P < 0.05).

αs2-Casein (%)12.1 ± 2.4b9.2 ± 2.4b29.2 ± 2.4a
αs1-Casein (%)39.5 ± 2.4a25.1 ± 2.4b5.9 ± 2.4c
β-Casein (%)37.2 ± 2.4b51.6 ± 2.4a50.5 ± 2.4a
κ-Casein (%)11.2 ± 2.413.8 ± 2.414.4 ± 2.4
Protein (%)90.94 ± 1.190.0 ± 1.190.0 ± 1.1
Ash (%)3.7 ± 0.33.9 ± 0.33.8 ± 0.3
Moisture (%)4.0 ± 0.24.2 ± 0.23.9 ± 0.2
Lactose (%)0.48 ± 0.030.49 ± 0.030.51 ± 0.03
image

Figure 1. Amino acid composition of bovine casein, caprine casein with high levels of αs1-casein and caprine casein with low levels of αs1-casein. The bovine casein values of the antioxidative amino acids histidine, methionine, phenylalanine, proline, tryptophan and tyrosine are significantly different than the two caprine values of such antioxidative amino acids (P < 0.05). There are no significant differences (P < 0.05) between bovine casein, caprine casein with high levels of αs1-casein and caprine casein with low levels of αs1-casein as to the amino acids targeted by mTG/glutamate (the sum of the target glutamine and glutamate) and lysine. mTG, microbial transglutaminase.

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The efficient production of high-quality emulsions depends on understanding the relationship between the bulk physicochemical characteristics and their colloidal properties. Emulsifiers, that is, casein and caseinates, have the ability to form and stabilise emulsions by being absorbed to the oil-in-water interface during homogenisation, reducing the interfacial tension by an appreciable amount, thus preventing droplet coalescence from occurring during homogenisation (Dickinson, 2003a). The minimum amount of an emulsifier required to produce a stable emulsion, its ability to produce small droplets during homogenisation and its ability to prevent small droplets from aggregating over time are the prerequisites for successful microencapsulation of fats and oils.

The caseins are adsorbed to an interface so that the predominantly nonpolar regions on the surface of the molecule face the oil phase, while the predominantly polar regions face the aqueous phase. The bovine and caprine caseins tend to have a particular orientation at an interface that depends strongly on their β-casein content (Mora-Gutierrez et al., 2010). Once absorbed to an interface, the bovine and caprine caseins may or may not undergo structural rearrangements, so that they can maximise the number of contacts between nonpolar groups and oil phase. Because of their hydrophobicity, high flexibility and their degree of absorption, the caseins are excellent emulsifying agents (Dickinson, 2003a). Consequently, the bovine and caprine casein aggregates, when inserted into the lipid droplets, become more compact as evidenced by 31P-NMR (Mora-Gutierrez et al., 2010). This ‘compactness’ was particularly more evident for the caprine casein aggregates. As mentioned earlier, the content of the phosphoprotein β-casein is high in caprine caseins (Table 1). Β-Casein has been found to form a dense interfacial layer surrounding oil droplets (Berton et al., 2011) as a result of inserting its hydrophobic site compactly into the oil droplets and presenting its hydrophilic end on the surface (Mora-Gutierrez et al., 2010). The 31P-NMR line widths and relaxation times of bovine and caprine caseins in algae oil-in-water emulsions (Mora-Gutierrez et al., 2010) also led us to the conclusion that at 0.5% (w/v), the bovine and caprine caseins are highly absorbed to the lipid droplets and that the maximal oil content is 5% with a protein ratio of 1:10. However, it has been found recently that in oil-in-water emulsions prepared with low amounts of proteins, that is, β-lactoglobulin, β-casein and bovine serum albumin (BSA) in the aqueous phase (0.5% w/v), the proteins adsorbed at the interface do not efficiently protect lipids against oxidation, in comparison with surfactants, that is, Tween-20, Tween-80 and CITREM (Berton et al., 2011). The protein-stabilised interfaces seem to be more heterogeneous and porous, while the surfactant-stabilised emulsions are more homogeneous and compact (Murray & Dickinson, 1996; Bos & van Vliet, 2001; Wilde et al., 2004). Nevertheless, a thick interfacial layer surrounding oil droplets has been shown to lower oxidation rates in emulsions emulsified with 0.2% (w/v) whey proteins (Hu et al., 2003a); whey proteins are a mixture of β-lactoglobulin, α-lactalbumin, lactoferrin, BSA and immunoglobulins. In a preliminary antioxidant efficacy testing of various concentrations of bovine and caprine caseins added to algae oil-in-water emulsions, it was found that 0.5% (w/v) casein provided antioxidant activity mainly by producing thick interfacial layers (Mora-Gutierrez et al., 2010). At these later concentrations, the bovine and caprine caseins (0.5% w/v) and the whey proteins (0.2% w/v) tend to form polymers (protein aggregates), which favour the interfacial phenomena, that is, the formation of a thick interfacial film around oil droplets, and may thus have contributed to the increase in oil stability (Hu et al., 2003a; Mora-Gutierrez et al., 2010). Moreover, in oil-in-water emulsions prepared with excess emulsifier (≥1.0% w/v), casein has been shown to be more effective in protecting against lipid oxidation than other proteins (Hu et al., 2003b; Faraji et al., 2004; Clausen et al., 2009; Frisenfeldt Horn et al., 2011). It is thought that the observed antioxidant property of caseins is related to the ability of the casein molecules to chelate metal ions (Faraji et al., 2004; Villiere et al., 2005; Sugiarto et al., 2010); to scavenge free radicals (Clausen et al., 2009); or to form thick interfacial layers (Fang & Dalgleish, 1993).

When the casein present in the two phases (lipid phase and aqueous phase) was separated by SDS-PAGE using MOPS running buffer and stained with Coomassie Brilliant Blue (Fig. 2), a similar electrophoretic pattern was shown in the lipid and aqueous phases of algae oil-in-water emulsions containing bovine casein or caprine casein high and low in αs1-casein (5%, w/v) after 48 h of incubation. On the other hand, when algae oil-in-water emulsions contained only 0.5% (w/v) casein (data not shown), very little protein was present in the aqueous phase; most of the protein was absorbed onto the lipid phase during incubation. No differences in the electrophoretic migration and/or casein distribution of the lipid and aqueous phases were shown in algae oil-in-water emulsions prepared with a higher concentration of bovine and caprine caseins characterised by high and low levels of αs1-casein.

image

Figure 2. SDS-PAGE of 5% caprine casein with high levels of αs1-casein in (a) solution (0% algae oil); (b) emulsion (10.5% algae oil) at t = 0 h; (c) emulsion (10.5% algae oil) at t = 48 h; (d) lipid phase of emulsion (10.5% algae oil) at t = 48 h; and (e) aqueous phase of emulsion (10.5% algae oil) at t = 48 h. The protein solution and emulsion samples contained 14.5% (w/v) maltodextrin (DE = 40). High pressure and temperature were applied to the protein solution and emulsion samples as described in ‘Methods’.

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Effect of mTG-cross-linked casein on emulsion stability

Enzymatic treatment of bovine casein with mTG is known to alter a number of functional properties, in particular emulsifying properties (Han & Damodaran, 1996). Therefore, the influence of mTG treatment at a temperature of 45 °C for 60 min on the emulsifying properties of bovine and caprine caseins was evaluated using EAI. The EAI is related to the surface area stabilised by a unit weight of proteins, which presents the ability of proteins to be absorbed at the interface of fat globules and the aqueous phase (Pearce & Kinsella, 1978). Table 2 shows the EAI values of control casein and mTG-modified casein emulsions. All the control casein emulsions from bovine and caprine origins exhibited no significant differences in EAI. However, the EAI values significantly (< 0.05) increased with enzyme treatments, and the EAI of caprine caseins with enzyme was greater (P < 0.05) than the EAI of bovine casein with enzyme. This may be attributed to the formation of low molecular mass cross-linked polymers, such as dimers and trimers, leading to a thin protein film at the oil-in-water interface. Molecular mass distributions of the modified bovine and caprine caseins formed during the 60-min enzyme exposure were analysed by SDS-PAGE (data not shown). Characterisation by SDS-PAGE indicates that the proportion of cross-linked polymers in caprine caseins was significantly (P < 0.05) more as compared to the bovine caseins. This is consistent with previous literature reports that β-casein is better accessible to mTG cross-linking than the αs-caseins (Dickinson & Yamamoto, 1996; Han & Damodaran, 1996). Due to the innate molten globule structure of the β-casein molecule (Farrell et al., 2006, 2013), glutamine and lysine residues are available on the surface of the molecule and may readily react with mTG. However, another reason could be that the caprine caseins have higher κ-casein content (Table 1) and mTG has been shown to produce multiple polymeric forms from κ-casein (Gembeh et al., 2005). Κ-Casein and its C-terminal hydrophilic peptide are also known to be surface oriented (Farrell et al., 2004, 2006) where they are susceptible to chymosin activity and mTG activity through glutamine residues 143 and 163 (Christensen et al., 1996). The extent of increase in EAI was not different in the mTG-modified casein emulsions prepared with the caprine caseins either from high or low αs1-casein source.

Table 2. Physical characteristics of the algae oil-in-water emulsions emulsified with casein and mTG-modified casein
CaseinEAI (m2 g−1)ESI[d3,2] (μm) at t = 0 h[d3,2] (μm) at t = 48 h
  1. EAI, emulsifying activity index; ESI, emulsifying stability index; mTG, microbial transglutaminase.

  2. Values represent the mean ± SD of triplicate measurements. Means in the same column with different letters a–c are different (P < 0.05).

Bovine16.5 ± 0.29c93.7 ± 2.2b0.25 ± 0.010.26 ± 0.01
Bovine + mTG23.3 ± 0.29b107.1 ± 2.2a0.25 ± 0.010.24 ± 0.01
Caprine high αs1-casein17.1 ± 0.29c98.0 ± 2.2b0.25 ± 0.010.24 ± 0.01
Caprine high αs1-casein + mTG24.5 ± 0.29a111.4 ± 2.2a0.26 ± 0.010.26 ± 0.01
Caprine low αs1-casein16.9 ± 0.29c97.6 ± 2.2b0.24 ± 0.010.25 ± 0.01
Caprine low αs1-casein + mTG24.4 ± 0.29a111.8 ± 2.2a0.23 ± 0.010.24 ± 0.01

The ability of an emulsifier to produce emulsions that remain stable to droplet aggregation can be measured by the emulsion stability index (ESI). The ESI was remarkably improved after the treatments with mTG (Table 2). The subtle differences in emulsion stability induced by mTG could reflect the differences in interactive forces involved in the formation of bovine and caprine milk-protein-stabilised oil droplets (Table 2). Hydrophobic interactions play a key role in emulsion stability, in particular in emulsions in which there is not enough emulsifier present to completely saturate the surfaces of the droplets (Dickinson, 2003b). A network of aggregated protein entrapping DHA oil droplets (exhibiting strong hydrophobic interactions) was described as the cause of the stability of emulsions prepared with 0.5% (w/v) bovine and caprine caseins (Mora-Gutierrez et al., 2010). However, steric interactions are considered the most common and important stabilising mechanism in food emulsions (Liu & Damodaran, 1999). To provide polymeric steric stabilisation, large amounts of emulsifier are needed to cover the droplet surface (because a thick interfacial layer is required). The relatively high values of ESI in emulsions treated with mTG (Table 2) may be attributed to the fact that the steric repulsion between absorbed protein layers was sufficiently strong to prevent droplet aggregation. Formation of a thicker layer of protruding cross-linked polymer chains by mTG would have been expected to increase the steric repulsion between the droplets, thereby increasing emulsion stability (Liu & Damodaran, 1999). In milk, κ-casein provides steric stabilisation for casein micelles (Farrell et al., 2004, 2006) through its hydrophilic C-terminal macropeptide; this should also add to the stabilisation of the emulsions (Dickinson, 2003a). In addition, caseins have a high net negative charge due to their high phosphoserine contents (Farrell et al., 2004); these residues are expected to occupy surface locations in emulsions (Dickinson, 2003a) and thus would also contribute to strong charge repulsions on these particles.

Emulsifier effectiveness at stabilising droplets against aggregation is often demonstrated by reporting the measured mean particle diameters or full particle distributions. The freshly prepared emulsions had very similar particle size during storage at 21 °C for 48 h, indicating that the emulsions were stable (Table 2). The particle size of the control casein emulsions did not differ from that of the mTG-modified casein emulsions during storage at 21 °C, and no phase separation (no creaming) was observed, showing that the emulsions were physically stable (Table 2).

Effect of antioxidant addition on emulsion stability

Lipid oxidation is generally caused by a radical reaction between unsaturated oils and oxygen. The lipid radicals are formed when loosely held hydrogen atoms are lost from the fatty acid group or excited double bonds. The resultant lipid radicals react with oxygen to form lipid peroxides. Lipid peroxides are primary oxidation products that can be transformed into various secondary products, including aldehydes, ketones, alcohols and hydrocarbons. Microencapsulation is an effective technique to delay lipid oxidation of oils rich in highly polyunsaturated fatty acids. The protective mechanism is to form a membrane (wall system) around oil droplets (core). The common method for microencapsulation is by spray-drying, due to low cost and readily available equipment (Goula & Adamopoulos, 2012). However, additional stabilisation with antioxidants is required to ensure maximum protection during processing and subsequent storage of microencapsulated oils rich in long-chain polyunsaturated fatty acids. In this context, it is generally accepted that the multiple physical environments and different operation steps during microencapsulation and subsequent storage require a balanced antioxidant composition different from the blends utilised for the stabilisation of bulk oils.

Certain spices contain phenolic compounds, which can delay the onset of oxidation. These compounds help neutralise the oxidation reaction by contributing hydrogen ions from their own hydroxyl groups to unstable free radicals that are formed during the initiation of oxidation. The phenolic compounds are still transformed into free radicals, but are more stable than the initial free radicals involved in oxidation. Thus, rather than preventing oxidation, they naturally slow its progression. Rosemary is one of the most widely used spices for its antioxidant properties. Among the active phenolic compounds are rosmarinic acid, carnosol and carnosic acid. The main active compounds carnosic acid and carnosol are considered to partition into the interface of the oil droplet (Hopia et al., 1996) and have shown high antioxidative activity in emulsified fish oil stabilised by tocopherols, ascorbyl palmitate and lecithin or CITREM (Pop, 2011). In this study, two oxidative parameters were evaluated: PV, to assess primary oxidation, and p-anisidine value (AV), to quantify secondary oxidation products in algae oil-in-water emulsions prepared with bovine and caprine caseins and their respective mTG-modified caseins. Understanding how a liposoluble rosemary extract rich in carnosic acid affects lipid oxidation of such casein-based emulsions could be used to improve the oxidative stability of algae oil microencapsulated with bovine and caprine caseins (the control) and mTG-modified bovine and caprine caseins (the treatment).

Many studies have shown that lipid oxidation rates are much dependent on the concentration of lipid hydroperoxides. The high pressure and temperature used to prepare milk emulsions fortified with fish oil resulted in a decrease in their oxidative stability (Sørensen et al., 2007). The caseins appeared to be the only milk proteins that oxidised at the beginning of the storage period (Sørensen et al., 2007). The addition of a ternary blend of lipophilic antioxidants, that is, tocopherols, ascorbyl palmitate, lecithin in combination with a liposoluble rosemary extract rich in carnosic acid, to fish oil led to a no increase in the hydroperoxide content during emulsion preparation and storage (Pop, 2011). In our experiments, the control caseins and mTG-modified caseins prevented the algae oil in the emulsions from oxidation during preparation and storage. Bovine and caprine caseins in the aqueous phase of algae oil emulsions increased oxidative stability of the algae oil due to the ability of caseins to act as a metal chelator in the aqueous phase, thereby preventing oxidation at the interface (Mora-Gutierrez et al., 2010). No significant differences (< 0.05) were observed in the PV or AV of the control caseins and mTG-modified caseins in the algae oil-in-water emulsions (Table 3), indicating no differences in oxidative stability of control and the treatments. Differences between the bovine and caprine caseins in oxidative stability are associated with differences in how these bovine and caprine milk proteins impact on the thickness or packing of the algae oil emulsion droplets (Mora-Gutierrez et al., 2010). A recent study indicated that β-casein formed a dense interfacial layer surrounding oil droplets (Berton et al., 2011), and β-casein is the most abundant protein in caprine caseins (Table 1). The lowest content of hydroperoxides was observed in casein-based, algae oil-in-water emulsion samples stabilised with liposoluble rosemary extract rich in carnosic acid during preparation and storage (Table 4). The liposoluble rosemary extract rich in carnosic acid may have had a protective effect on the algae oil by working synergistically with the tocopherol isomer δ. The relatively low levels of the tocopherol isomer α may contribute to the oxidative stability of algae oil in the casein-based emulsions. Our findings are in agreement with the work conducted by Pop (2011), where the oxidative stabilisation of fish oil during emulsion preparation, encapsulation and storage required a combination of tocopherols high in the isomer δ and low in the isomer α and liposoluble rosemary extract rich in carnosic acid. Generally, a pro-oxidant effect of α-tocopherol occurs when it is present in relatively high concentrations (Nawar, 1996). In this study, the combined action of a metal chelator such as the bovine and caprine caseins, a free radical acceptor such as δ-tocopherol and a free radical scavenger such as the phenolic compounds in rosemary extracts (e.g. carnosic acid, carnosol) may work synergistically to protect algae oil in oil-in-water emulsions. The development of secondary oxidation products as measured by AV was also lowest in casein-based, algae oil-in-water emulsion samples stabilised with liposoluble rosemary extract rich in carnosic acid during preparation and storage (Table 4).

Table 3. Peroxide and p-anisidine values in algae oil-in-water emulsions prepared with casein-based emulsifiers stored at 2 °C
Casein-based emulsifierPeroxide value (mmol L1)p-Anisidine value
at t = 0 hat t = 48 hSEMat t = 0 hat t = 48 hSEM
  1. mTG, Microbial transglutaminase.

  2. Values represent the mean ± SD of triplicate measurements. The means within each column are not significantly different (P < 0.05).

Bovine casein0.0700.0720.0041.001.070.19
Bovine casein + mTG0.0670.0680.0040.981.000.19
Caprine high αs1-casein0.0630.0650.0040.981.000.19
Caprine high αs1-casein + mTG0.0620.0630.0040.960.970.19
Caprine low αs1-casein0.0610.0630.0040.981.000.19
Caprine low αs1-casein + mTG0.0580.0620.0040.960.970.19
Table 4. Peroxide and p-anisidine values in algae oil-in-water emulsions prepared with casein-based emulsifiers stabilised with liposoluble rosemary extract rich in carnosic acid stored at 2 °C
Casein-based emulsifierPeroxide value (mmol L1)p-Anisidine value
at t = 0 hat t = 48 hSEMat t = 0 hat t = 48 hSEM
  1. mTG, Microbial transglutaminase.

  2. Values represent the mean ± SD of triplicate measurements. The means within each column are not significantly different (P < 0.05).

Bovine casein0.0610.0620.0050.490.510.086
Bovine casein + mTG0.0570.0580.0050.480.50.086
Caprine high αs1-casein0.0500.0550.0050.330.370.086
Caprine high αs1-casein + mTG0.0490.0520.0050.340.360.086
Caprine low αs1-casein0.0560.0570.0050.330.340.086
Caprine low αs1-casein + mTG0.0510.0530.0050.320.330.086

Effect of lipid–protein interactions on emulsion stability

Interaction between lipids and proteins also affects the quality of fish oil-in-water emulsions due to the development of off-flavours caused by oxidation of specific amino acids (Frisenfeldt Horn et al., 2011) Our data show the changes in the fluorescence emission spectrum of caprine casein with low levels of αs1-casein treated with mTG in the presence of increasing molar ratios of carnosic acid (Fig. 3). When an algae oil-in-water emulsion (without phenols) emulsified with lecithin and caprine casein with low levels of αs1-casein treated with mTG was excited as its absorption maximum of 280 nm, a fluorescence emission spectrum with a maximum at 350 nm was observed (uppermost line in Fig. 3). Addition of increasing molar ratios of carnosic acid to the caprine casein low in αs1-casein treated with mTG resulted in a progressive quenching of the fluorescence emission. The results are suggestive of binding of carnosic acid to the caprine casein with low levels of αs1-casein treated with mTG. This peak was quenched by the increasing molar ratios of carnosic acid in the same pattern as that observed with caprine casein with low levels of α1-casein treated with mTG (Fig. 3) in all the control caseins and mTG-modified caseins used in this study (data not shown). These results lead to the following conclusions: (i) carnosic acid is capable of binding to caseins; (ii) at least one of the modes of binding of carnosic acid to caseins involves tryptophan residues; and (iii) the tryptophan residues are located in an hydrophilic environment, that is, on the surface of the protein molecule and thus exposed to protein–lipid and other interaction reactions (Burstein et al., 1973). The shielding of tryptophan in the control and mTG-modified casein samples by carnosic acid may protect this residue from further oxidation in algae oil-in-water emulsions. Carnosic acid, in addition to tryptophan, may also bind to other amino acids (e.g. histidine and methionine).

image

Figure 3. Effect of increasing concentrations of carnosic acid on the fluorescence emission spectra of mTG-modified caprine casein with low levels of αs1-casein in algae oil emulsions emulsified with lecithin. mTG-modified caprine casein with low levels of αs1-casein was excited at 280 nm, and the emission spectra were recorded between 300 and 400 nm. Traces, from top to bottom, are mTG-modified caprine casein with low levels of αs1-casein alone (0.1 μm); mTG-modified caprine casein with low levels of αs1-casein/carnosic acid molar ratios of 1:0.1, 1:0.2, 1:0.4, 1:0.6, 1:0.8; 1:1, 1:1.2, 1:2.5, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:10, 1:11, 1:12, 1:13, 1:14 and 1:15, respectively. Note that at ratios above 1:3, the observed quenching is due primarily to inner filter effects from the carnosic acid and not binding (Brand & Witholt, 1967). mTG, microbial transglutaminase.

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The homogenisation conditions used in this study did not significantly influence the development of protein carbonyls, which is one of the products formed during protein oxidation (Table 5). However, the results showed that liposoluble rosemary extract rich in carnosic acid actually inhibited protein oxidation initiated by homogenisation of the algae oil emulsified with the control caseins and mTG-modified caseins as inferred by the level of carbonyl groups, which are introduced as a result of protein oxidation (Table 5). Development of protein carbonyls during the storage of the casein-based, algae oil-in-water emulsions stabilised with liposoluble rosemary extract rich in carnosic acid was significantly lower (Table 5).

Table 5. Formation of protein carbonyl compounds in algae oil-in-water emulsions prepared with casein-based emulsifiers in the absence and presence of liposoluble rosemary extract rich in carnosic acid stored at 2 °C
Casein-based emulsifierWithout rosemaryWith rosemary
at t = 0 hat t = 48 hSEMat t = 0 hat t = 48 hSEM
  1. mTG, Microbial transglutaminase.

  2. Values represent the mean ± SD of triplicate measurements. Means within each column with different superscripts are significantly different (P < 0.05).

Bovine casein1.101.180.170.58a0.640.005
Bovine casein + mTG1.071.200.170.56a0.630.005
Caprine high αs1-casein1.131.170.170.42b0.640.005
Caprine high αs1-casein + mTG1.101.170.170.40b0.660.005
Caprine low αs1-casein1.071.200.170.41b0.680.005
Caprine low αs1-casein + mTG1.031.130.170.38b0.670.005

Effect of polyphenol–protein interactions on emulsion stability

Interaction of plant polyphenols (PPs) with proteins appears to be important in the stabilising effect of such PPs. The ability of PP to interact with milk proteins is established (Roginsky & Alegria, 2005). It was reported that PPs bound to milk proteins are less accessible for oxidation than free PPs (Roginsky & Alegria, 2005). The addition of PP to milk and whey proteins resulted in a reduction in lysine and tryptophan (O'Connell & Fox, 1999; Rawel et al., 2001). The whey protein derivatives formed by covalent bonding with PPs influenced the hydrophobic–hydrophilic character of the whey proteins (Rawel et al., 2001). It is speculated that the lower amount of lysine and tryptophan available for reaction with carnosic acid and carnosol present in the rosemary extract used in this study may have less influence on the hydrophobic–hydrophilic character of the caprine caseins (Table 1), which translates into a greater affinity of the caprine caseins for the algae oil and a greater antioxidative effect of the absorbed caprine caseins. Comparison of data between Tables 3 and 4 indicates that the antioxidant properties of the bovine and caprine caseins in the algae oil-in-water emulsions were positively affected by the addition of liposoluble rosemary extract rich in carnosic acid. Caprine caseins did not show any significant difference when compared with the bovine casein in terms of antioxidant property, but in the presence of caprine caseins, the reduction in PV and AV was slightly lower (Table 4). According to the above PV, AV and protein stability results, it is evident that the addition of liposoluble rosemary extract rich in carnosic acid to the casein-based, algae oil-in-water emulsions has positive effects on maintaining the ‘freshness’ of such emulsions during the storage. Therefore, these emulsions were used for microencapsulation.

Effect of microencapsulation on powder stability

Microencapsulation of fats and oils with milk proteins requires not only satisfying the emulsifying effectiveness, but also minimising the amount of free fat or oil (and covering the fishy flavour and/or odour of the fats and oils) in the system. Microencapsulation of fats and oils is optimised by the formation of small emulsion droplets. For this purpose, enough emulsifier must be present to confer interfacial stability to emulsions, as evidenced in this study by the consistently smaller average droplet sizes (Table 2). Microencapsulation efficiency of algae oil by the bovine and caprine caseins previously treated with mTG was ≥ 97% (Table 6). This means that the amount of free DHA oil was negligible in the system. In the cases of caprine caseins with high and low levels of αs1-casein, a somewhat more compact wall film is observed due to effective covalent cross-linking by mTG. As a result, lower PV values are observed for the two caprine casein powders immediately after spray-drying (Table 6).

Table 6. Microencapsulation efficiency and initial peroxide value of spray-dried algae oil-in-water emulsions microencapsulated with casein and mTG-modified casein with added liposoluble rosemary extract rich in carnosic acid and δ-tocopherol
CaseinMicroencapsulation efficiency (%)Peroxide value (mmol kg1)
  1. mTG, Microbial transglutaminase.

  2. Values represent the mean ± SD of triplicate measurements. Means in the same column with different letters a–d are different (P < 0.05).

Bovine93.8 ± 0.99b0.77 ± 0.2a
Bovine + mTG97.5 ± 0.99a0.50 ± 0.2b
Caprine high αs1-casein95.0 ± 0.99b0.73 ± 0.2a
Caprine high αs1-casein + mTG99.3 ± 0.99a0.30 ± 0.2d
Caprine low αs1-casein95.4 ± 0.99b0.73 ± 0.2a
Caprine low αs1-casein + mTG99.1 ± 0.99a0.40 ± 0.2c

The high ME of bovine and caprine casein powders prepared by using mTG indicate the formation of wall matrices capable of conferring high levels of protection to microencapsulated algae oil against lipid oxidation during storage for 4 weeks at 30 °C and 33% relative humidity (Table 7). The PV of controls and each mTG treatments significantly increased for each week of storage as the storage period progressed (Table 7). In the same table, it can readily be seen that the mTG treatments significantly lowered the amount of peroxides generated during the storage period. The mTG-modified caprine caseins are also notably more effective than their bovine counterpart.

Table 7. Peroxide values (mmol kg−1) for spray-dried algae oil-in-water emulsions microencapsulated with casein and mTG-modified casein with added liposoluble rosemary extract rich in carnosic acid and δ-tocopherol
CaseinTime (weeks)SEM
01234
  1. mTG, Microbial transglutaminase.

  2. Values represent the mean ± SD of triplicate measurements. Means in the same columns with different letters a–e are different (P < 0.05). Means in the same rows with different letters f–j are different (P < 0.05).

Bovine0.77f4.93ag6.77ah10.63ai13.01aj0.2
Bovine + mTG0.50f2.90bg3.47dh5.80ci8.60cj0.2
Caprine high αs1-casein0.73f3.23bg5.40ch7.93bi9.53bj0.2
Caprine high αs1-casein + mTG0.30f1.50cg2.33eh4.16di6.23dj0.2
Caprine low αs1-casein0.73f3.37bg6.03bh8.30bi9.80bj0.2
Caprine low αs1-casein + mTG0.40f1.37cg2.06eh3.87di5.67ej0.2

Lipid oxidation leads to a reduction in the concentration of health-promoting n-3 fatty acids, as well as to the generation of volatile compounds that may cause an undesirable rancid flavour. The improved oxidative stability of algae oil microencapsulated in bovine and caprine casein-based wall matrices induced by mTG is consistent with the findings of Bao et al. (2011), using bovine sodium caseinate matrices cross-linked by mTG to stabilise algae oil against lipid oxidation. The enhanced oxidative stability of the microencapsulated algae oil has been ascribed to the oxygen permeability barrier properties of bovine-sodium-caseinate-based wall matrices induced by mTG (Bao et al., 2011). Indeed, oxygen permeability is a determining factor of the oxidative stability of microcapsules (Drusch & Mannino, 2009). A compact network within the caprine casein films may be formed due to the effective covalent cross-links by mTG (Table 7). Thus, a decrease in hydroperoxide contents in spray-dried algae oil emulsions microencapsulated with bovine and caprine casein-based matrices was significantly (P < 0.05) correlated with the dense network formed as a result of the cross-linking reaction by mTG (Table 7), all of which reduce the permeability of oxygen through the protein network as well as extend the shelf life of the algae oil. The formation of secondary oxidation products, which has a high impact on off-flavour, was also significantly low (P < 0.05) in algae oil microencapsulated with the mTG-modified bovine and caprine caseins during storage (Table 8). The amount of secondary oxidation products detected in the microencapsulated algae oil was significantly (P < 0.05) affected by the wall system variable (Table 8). Thus, major differences between the wall matrices of bovine and caprine caseins were observed with the caprine casein wall systems being more effective.

Table 8. p-Anisidine value for spray-dried algae oil-in-water emulsions microencapsulated with casein-based wall matrices with added liposoluble rosemary extract rich in carnosic acid and δ-tocopherol stored for 4 weeks at 30 °C and 33% relative humidity
Casein-based wall matrixp-Anisidine valueSEM
  1. Values represent the mean ± SD of triplicate measurements. Means with different superscripts are significantly different (P < 0.05).

Bovine casein15.17a0.45
Bovine casein + mTG12.23b0.45
Caprine high casein9.90c0.45
Caprine high casein + mTG7.70d0.45
Caprine low casein9.77c0.45
Caprine low casein + mTG7.43d0.45

Although we used the same microencapsulating approach of Bao et al. (2011) to effectively stabilise algae oil against oxidation, that is, mTG cross-linking of sodium caseinate using a cross-linking time of 30 min at 45 °C, direct comparison of the PV generated in this study with those of Bao et al. (2011) is difficult due to variation in the nature of the systems examined: (i) the bovine and caprine caseins are presented in this study as molecular clusters of potassium caseinates; (ii) the free glucose present at higher levels in maltodextrin DE = 40 (this study) could (a) hydrogen bond with hydroperoxides, resulting in higher antioxidant effect (Anandaraman & Reineccius, 1986), (b) the improved oxygen barrier properties of high DE maltodextrin DE = 40 (Anandaraman & Reineccius, 1986) and/or the enhanced hydration of the potassium caseinate films conferred by the high DE maltodextrin during the process of spray-drying, which increases the stability of such potassium caseinate films leading to less DHA oil leaking out of the power surface (Fäldt & Bergenståhl, 1995; Mora-Gutierrez et al., 1997); (iii) the higher levels of Maillard reaction products are formed from bovine casein or caprine caseins with high and low levels of α1-casein and glucose with antioxidant activity (McGookin & Augustine, 1991); and (iv) the addition to the emulsions of a liposoluble rosemary extract rich in carnosic acid has been shown to inhibit oxidation of long-chain n-3 fatty acids (e.g. fish oil) in emulsions and microcapsules (Pop, 2011).

Sensory studies are needed to determine whether the developed algae oil microcapsules are sensory acceptable during 24 months of storage at temperatures of 5 and 30 °C and relative humidities of 15%, 25% and 33%.

Conclusions

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgments
  8. References

Oxidative stability of algae oil in oil-in-water emulsions emulsified with bovine and caprine caseins and mTG-modified caseins can be successfully achieved with the addition of rosemary extract rich in carnosic acid in combination with tocopherols high in the δ-isomer and low in the α-isomer. Because of their innate molten globule structures (Farrell et al., 2013), the caseins are accessible towards oxidative changes. Such free radical damage to the caseins might play a significant role in the propagation reaction of lipid oxidation. The data presented here confirm that phenolic compounds (e.g. carnosic acid and carnesol in rosemary extracts) can act as antioxidants by retarding lipid/casein oxidation or by binding to caseins. The spectrofluorometric evidence for the binding of carnosic acid to the caseins relates to some nonspecific binding and, in part, to changes in fluorescence of tryptophan residues. The spray-dried powders produced from the bovine- and caprine-based emulsions stabilised with liposoluble rosemary extract rich in carnosic acid exhibited a high stability upon storage, indicating that the level of hydroperoxides in liquid emulsions has a strong influence on the shelf life of the spray-dried powders. Caprine caseins were found to be the most effective wall systems in spray-dried powders. The present study provides useful information for protecting lipid oxidation, especially algae oil oxidation, in liquid and dried emulsion systems.

Acknowledgments

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgments
  8. References

This work was supported by Evans-Allen funding through the United States Department of Agriculture/Cooperative State Research Service.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgments
  8. References
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