**3. Results and discussion**

The rate and extent of oxidation of marine oils i.e., algae oil, fish oil depends on the matrix of the food to be fortified. Milk relative to some other foods offers good protection against oxidation, since these marine oils are emulsified and stabilized by the casein micellar structure [18]. Casein adsorbs to the newly formed interface thereby, providing enhanced protection by forming a physical barrier [31, 32]. Although milk is stored in refrigerators (2–4°C) and has a relatively short life (21 days), it is still subject to overall stress due to UV and visible light, temperature fluctuations, and handling abuse.

Lipid oxidation proceeds from the interface to the oil droplet interior in oil-inwater emulsions i.e., goat milk emulsion, therefore, the susceptibility of lipids to oxidation at the interface is the most important factor affecting the oxidative stability of lipids in food and beverage emulsions. It is generally accepted that the attack of free radicals and trace metals on lipids at the interface increases with the increase in the area of interface. Thus, the oxidative stability of DHA in goat milk emulsions should decrease with decreasing droplet sizes. The results of the droplet size determinations (**Table 2**) showed that the droplets did not change in size from day 1 to day 14 of storage at 2°C, indicating that the goat milk emulsions were physically stable during the 2 weeks of storage. The average droplet size in all goat milk emulsions containing 0.5% oil was from 1.20 ± 0.01 to 1.25 ± 0.03 μm, while the droplet size in the original goat milk sample was 0.89 ± 0.02 μm. These results showed that the sizes of droplets in goat milk emulsions containing 0.5% added oil, irrespective of the oil type, were significantly (p < 0.05) larger than the droplets in the original goat milk sample. The decrease in the oil droplet size induces the increase in the droplet interface [33], from which the oxidation proceeds to the oil droplet interior.


**Table 2.**

**119**

**Figure 2.**

*significantly different (p < 0.05).*

*Phenolic Compounds in Hibiscus mutabilis Seeds and Their Effects on the Oxidative Stability…*

The results of **Table 2** suggest that DHA in goat milk emulsions containing 0.5%

As to the goat milk emulsions, the chromatographically purified *Hibiscus mutabilis* seed oil (PHMO) together with the chromatographically purified algae oil (PAO) had a significantly (p < 0.05) higher PV than the other two goat milk emulsions at each storage time at 2°C (**Figure 2**). On the other hand, the goat milk emulsion with the natural *Hibiscus mutabilis* seed oil (NHMO) and the chromatographically purified algae oil (PAO) exhibited good oxidative stability as inferred from a low PV (**Figure 2**). The goat milk emulsion containing the chromatographically purified algae oil (PAO) and the natural *Hibiscus mutabilis* seed oil (NHMO) with added ascorbyl palmitate (LAAP) had the lowest PV during the study (**Figure 2**). These results suggest that the presence of antioxidants i.e., γ-tocopherol, ascorbyl palmitate improved the oxidative stability of goat milk emulsions under storage at 2°C for 14 days, which may contribute to the

The key challenge in formulating food products with marine oils is their sensitivity to iron and copper, catalysts to oxidation that exists in even the cleanest water, foods, and other ingredients. The goat milk used in this study, contained approximately 138 ppb iron and 27 ppb copper, and these values were not affected by the addition of oils to the goat milk. The presence of trace metals in goat milk

*Peroxide values of goat milk emulsions containing the different oils with and without added ascorbyl palmitate during 14-day storage at 2°C. Means (n = 3) within each storage day with different letters (a–c) are* 

When supplementing foods with algae oil or fish oil, it is important to consider the initial quality of the raw material. In this work, we have ensured that all oils tested had similar variables such as age and storage temperature. The results of **Table 1** showed that all oils had low initial values of PV, and very low content of free fatty acids. Rancid flavors in goat milk is usually associated to the release of short-chain free fatty acids and is a persistent problem in dairy goat farms due to mishandling procedures starting from the farm until it reaches the consumers. The content of free fatty acids in the goat milk used in this study was very low (0.1%) and likewise, the PV content of goat milk was also low (0.1 meq/kg) suggesting that the quality of goat milk was

added oil is expected to be less oxidized in the droplet interior.

*DOI: http://dx.doi.org/10.5772/intechopen.80541*

acceptable from the sensory perspectives.

shelf life of goat milk emulsions.

*Droplet sizes of goat milk emulsions prepared by mixing chromatographically purified algae oil (0.25 wt%), chromatographically purified Hibiscus mutabilis seed oil (0.25 wt%) or natural Hibiscus mutabilis seed oil (0.25 wt%) with and without added ascorbyl palmitate (200 μg/g of oil) during 14-day storage at 2°C.*

#### *Phenolic Compounds in Hibiscus mutabilis Seeds and Their Effects on the Oxidative Stability… DOI: http://dx.doi.org/10.5772/intechopen.80541*

The results of **Table 2** suggest that DHA in goat milk emulsions containing 0.5% added oil is expected to be less oxidized in the droplet interior.

When supplementing foods with algae oil or fish oil, it is important to consider the initial quality of the raw material. In this work, we have ensured that all oils tested had similar variables such as age and storage temperature. The results of **Table 1** showed that all oils had low initial values of PV, and very low content of free fatty acids. Rancid flavors in goat milk is usually associated to the release of short-chain free fatty acids and is a persistent problem in dairy goat farms due to mishandling procedures starting from the farm until it reaches the consumers. The content of free fatty acids in the goat milk used in this study was very low (0.1%) and likewise, the PV content of goat milk was also low (0.1 meq/kg) suggesting that the quality of goat milk was acceptable from the sensory perspectives.

As to the goat milk emulsions, the chromatographically purified *Hibiscus mutabilis* seed oil (PHMO) together with the chromatographically purified algae oil (PAO) had a significantly (p < 0.05) higher PV than the other two goat milk emulsions at each storage time at 2°C (**Figure 2**). On the other hand, the goat milk emulsion with the natural *Hibiscus mutabilis* seed oil (NHMO) and the chromatographically purified algae oil (PAO) exhibited good oxidative stability as inferred from a low PV (**Figure 2**). The goat milk emulsion containing the chromatographically purified algae oil (PAO) and the natural *Hibiscus mutabilis* seed oil (NHMO) with added ascorbyl palmitate (LAAP) had the lowest PV during the study (**Figure 2**). These results suggest that the presence of antioxidants i.e., γ-tocopherol, ascorbyl palmitate improved the oxidative stability of goat milk emulsions under storage at 2°C for 14 days, which may contribute to the shelf life of goat milk emulsions.

The key challenge in formulating food products with marine oils is their sensitivity to iron and copper, catalysts to oxidation that exists in even the cleanest water, foods, and other ingredients. The goat milk used in this study, contained approximately 138 ppb iron and 27 ppb copper, and these values were not affected by the addition of oils to the goat milk. The presence of trace metals in goat milk

#### **Figure 2.**

*Biochemistry and Health Benefits of Fatty Acids*

The results of triplicate analyses were expressed as means ± standard deviations. The data were analyzed by ANOVA using PRO GLM procedure of SAS (version 8.2, SAS Institute, Cary, NC, USA). The least significant difference test was used to deter-

The rate and extent of oxidation of marine oils i.e., algae oil, fish oil depends on the matrix of the food to be fortified. Milk relative to some other foods offers good protection against oxidation, since these marine oils are emulsified and stabilized by the casein micellar structure [18]. Casein adsorbs to the newly formed interface thereby, providing enhanced protection by forming a physical barrier [31, 32]. Although milk is stored in refrigerators (2–4°C) and has a relatively short life (21 days), it is still subject to overall stress due to UV and visible light, temperature

Lipid oxidation proceeds from the interface to the oil droplet interior in oil-inwater emulsions i.e., goat milk emulsion, therefore, the susceptibility of lipids to oxidation at the interface is the most important factor affecting the oxidative stability of lipids in food and beverage emulsions. It is generally accepted that the attack of free radicals and trace metals on lipids at the interface increases with the increase in the area of interface. Thus, the oxidative stability of DHA in goat milk emulsions should decrease with decreasing droplet sizes. The results of the droplet size determinations (**Table 2**) showed that the droplets did not change in size from day 1 to day 14 of storage at 2°C, indicating that the goat milk emulsions were physically stable during the 2 weeks of storage. The average droplet size in all goat milk emulsions containing 0.5% oil was from 1.20 ± 0.01 to 1.25 ± 0.03 μm, while the droplet size in the original goat milk sample was 0.89 ± 0.02 μm. These results showed that the sizes of droplets in goat milk emulsions containing 0.5% added oil, irrespective of the oil type, were significantly (p < 0.05) larger than the droplets in the original goat milk sample. The decrease in the oil droplet size induces the increase in the droplet interface [33], from which the oxidation proceeds to the oil droplet interior.

*Droplet sizes of goat milk emulsions prepared by mixing chromatographically purified algae oil (0.25 wt%), chromatographically purified Hibiscus mutabilis seed oil (0.25 wt%) or natural Hibiscus mutabilis seed oil (0.25 wt%) with and without added ascorbyl palmitate (200 μg/g of oil) during 14-day storage at 2°C.*

mine significant differences among treatment means at p < 0.05.

**2.8 Statistical analysis**

**3. Results and discussion**

fluctuations, and handling abuse.

**118**

**Table 2.**

*Peroxide values of goat milk emulsions containing the different oils with and without added ascorbyl palmitate during 14-day storage at 2°C. Means (n = 3) within each storage day with different letters (a–c) are significantly different (p < 0.05).*

is expected to accelerate the degradation of lipid hydroperoxides as well as the degradation of the secondary oxidation products into shorter chain volatiles.

The results of **Figure 3** showed that the goat milk emulsions containing a mixture (1:1) of the chromatographically purified algae oil (PAO) and the chromatographically purified *Hibiscus mutabilis* seed oil (PHMO) were more oxidized than the goat milk emulsions containing a mixture (1:1) of the chromatographically purified algae oil (PAO) and the natural *Hibiscus mutabilis* seed oil (NHMO). The protective effect of the natural *Hibiscus mutabilis* seed oil (NHMO) may be partially ascribed to the high content of tocopherols, especially γ-tocopherol. As pointed out earlier, the tocopherols are free-radical terminators, which donate a hydrogen to the peroxyl radical [12]. Goat milk contains citric acid [34], and citric acid is recognized as a metal chelator. The chelating properties of citric acid have been proposed to protect tocopherols during oxidation [35]. Therefore, citric acid in goat milk could enhance the antioxidant activity of tocopherols in the emulsions containing the natural *Hibiscus mutabilis* seed oil (NHMO) and the chromatographically purified algae oil (PAO) at 1:1 ratio (**Figure 3**).

Ascorbyl palmitate (LAAP), which was added (200 μg/g of oil) to the natural *Hibiscus mutabilis* seed oil (NHMO) and the chromatographically purified algae oil (PAO) at 1:1 ratio, significantly reduced (p < 0.05) the extent of oxidation in this goat milk emulsion at 7-day and 14-day storage at 2°C (**Figure 3**). This protective effect of added ascorbyl palmitate (LAAP) was not observed in goat milk emulsions containing the chromatographically purified *Hibiscus mutabilis* seed oil (PHMO) and the chromatographically purified algae oil (PAO) at 1:1 ratio during 14-day storage at 2°C (data not shown). Ascorbyl palmitate (LAAP) had a more pronounced protective effect on the goat milk emulsion prepared with the chromatographically purified algae oil (PAO) and the natural *Hibiscus mutabilis* seed oil (NHMO) at 1:1 ratio by working synergistically with the γ-tocopherol isomer at 7-day and 14-day storage at 2°C (**Figure 3**). It is likely that ascorbyl palmitate retarded oxidation during storage of oil-in-water emulsions by direct scavenging of free radicals and tocopherol regeneration [18].

#### **Figure 3.**

*p-Anisidine values of goat milk emulsions containing the different oils with and without added ascorbyl palmitate during 14-day storage at 2°C. Means (n = 3) within each storage day with different letters (a–c) are significantly different (p < 0.05).*

**121**

*Phenolic Compounds in Hibiscus mutabilis Seeds and Their Effects on the Oxidative Stability…*

There is a direct relationship between the level of oxidation and sensory deterioration and even at times when there are no detectable oxidation parameters, the taste of the finished products could be displeasing. The decomposition of lipid hydroperoxides from marine derived *n*-3 polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA, C22:6*n*-3) and eicosapentaenoic acid (EPA, C20:5*n*-3), produces undesirable rancid and fishy off-flavors. Future work will evaluate the sensory attributes of DHA-enriched goat milk emulsion stabilized by

This work showed the suitability of using *Hibiscus mutabilis* seed oil to protect marine-derived *n*-3 PUFAs in oil-in-water emulsions i.e., DHA-milk from oxidative degradation for 14 days at 2°C. The natural *Hibiscus mutabilis* seed oil efficiently protected the chromatographically purified algae oil from oxidation during emulsification and storage of DHA-enriched goat milk emulsion. The addition of ascorbyl palmitate to the natural *Hibiscus mutabilis* seed oil and the chromatographically purified algae oil prior to goat milk emulsification had a significant (p < 0.05) protective effect on DHA-enriched goat milk emulsion. The combination of differences in fatty acid composition and concentration of tocopherols for the natural *Hibiscus mutabilis* seed oil seems to affect the oxidative stability of the goat milk emulsions prepared with this oil. This study provides a useful precedent for understanding the antioxidant activity of *Hibiscus* seed oils in food and beverage emulsions containing

Complementary work is currently being performed in our laboratory to optimize the oxidative stability of DHA-enriched goat milk emulsions with added seed oils from *Hibiscus* species such as *Hibiscus moscheutos* and *Hibiscus dasycalyx* to be able to withstand the thermal and mechanical stresses of industrial processes.

This work was supported by Evans-Allen funding to the Cooperative Agricultural Research Center through USDA Cooperative State Research Service.

The authors declare that they have no conflict of interest.

*DOI: http://dx.doi.org/10.5772/intechopen.80541*

**4. Conclusions**

marine *n*-3 PUFAs.

**Acknowledgments**

**Conflict of interest**

the natural *Hibiscus mutabilis* seed oil and ascorbyl palmitate.

*Phenolic Compounds in Hibiscus mutabilis Seeds and Their Effects on the Oxidative Stability… DOI: http://dx.doi.org/10.5772/intechopen.80541*

There is a direct relationship between the level of oxidation and sensory deterioration and even at times when there are no detectable oxidation parameters, the taste of the finished products could be displeasing. The decomposition of lipid hydroperoxides from marine derived *n*-3 polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA, C22:6*n*-3) and eicosapentaenoic acid (EPA, C20:5*n*-3), produces undesirable rancid and fishy off-flavors. Future work will evaluate the sensory attributes of DHA-enriched goat milk emulsion stabilized by the natural *Hibiscus mutabilis* seed oil and ascorbyl palmitate.
