**1. Introduction**

Human breast milk contains both docosahexaenoic acid (DHA, C22:6*n*-3) and arachidonic acid (AA, C20:4*n*-6) [1], which are essential for health. Studies in animals and humans indicate that DHA is essential for normal visual and brain function in the premature infants and possibly in the full term infants [2, 3]. In some breast-fed infants, colic has been related to the mother's consumption of cow milk [4, 5]. In older infants, the incidence of cow milk protein intolerance was encountered in 5–15% of cases [6]. A popular therapy among pediatricians is to change from cow milk to vegetable protein soy-based formula; however, infants with cow milk protein intolerance will also react adversely to soybean proteins [7]. When the problem is allergy to cow milk proteins (casein, whey), goat milk is a suitable substitute to cow milk [8].

The omega-3 fatty acids in the milk of grass-fed goats are predominantly linolenic acid (C18:3*n*-3), or alpha (α)-linolenic acid (ALA). DHA can be synthesized from dietary ALA, but the human body can only make very small amounts of DHA from ALA [9]. Therefore, there is a need to supplement foods with DHA. The addition of DHA from algae oil in food emulsions such as in goat milk emulsion requires the need for antioxidants. Antioxidants increase the shelf life of emulsions, but a clean label ingredient is required when added to milk. Oxidation in oil-in-water emulsions is thought to occur at the interface region between the oil and the aqueous phases [10]. In the oil phase of the emulsions, fatty acids are the target of free radicals i.e., hydroxyl radicals, which stimulate lipid peroxidation. Nonpolar antioxidants such as tocopherols and ascorbyl palmitate have been shown to be highly effective in protecting oil-in-water emulsions [11]. Tocopherols are free-radical terminators thereby, interrupting the free-radical chain of oxidative reactions by contributing hydrogen from the phenolic hydroxyl groups [12]. Ascorbyl palmitate, a lipid-soluble antioxidant, exhibits antioxidant activities that include single oxygen quenching and free-radical scavenging [13–15]. Ascorbyl palmitate has been shown to work synergistically with tocopherols by donating a hydrogen to the tocopheroxyl radical, formed as a result of tocopherol donating a hydrogen to the lipid radical [16, 17].

The oxidative stability of DHA-enriched emulsion may also be accomplished by the addition of vegetable oil to algae oil. In this context, one of the strategies developed to protect fish oil in a cow milk emulsion against oxidation was the mixing of rapeseed oil with fish oil prior to emulsification of cow milk [18]. The authors found that tocopherol isomers in concentrations similar to those found in natural rapeseed oil, and added to rapeseed oil stripped of natural tocopherols, significantly inhibited oxidation in cow milk emulsions enriched with fish oil [18].

*Hibiscus mutabilis* (Malvaceae) are shrubs with peach color flowers and originally native of China. The seeds of *Hibiscus mutabilis*, which do not have economic applications yet, are a source of vegetable oil. Although not widely reported in the literature, a high content of phenolic compounds, tocopherols are found in *Hibiscus mutabilis* seed oil. The seeds of *Hibiscus mutabilis* are also a source of lectin. Lectin from the seeds of *Hibiscus mutabilis* has carbohydrate-binding specificity to galactonic acid, which potently inhibited HIV-1 reverse transcriptase [19]. HIV, the RNA virus that causes AIDS, gradually disrupts the immune system in humans. Since a recent study suggested that DHA in high DHA-concentrated fish oil positively contributed to certain aspects of immune function in middle-aged obese adults [20], DHA-enriched goat milk stabilized by *Hibiscus mutabilis* seed oil potentially can be used as immune stimulator for the adjunctive therapy of HIV.

In the present work the suitability of *Hibiscus mutabilis* seed oil for enhancing the oxidative stability of DHA-enriched goat milk emulsion was studied. Based on the potential synergistic effects of ascorbyl palmitate with tocopherols, it was assumed that the most efficient oxidative stabilization during homogenization and storage of DHA-enriched goat milk may be achieved by combining both of these lipophilic antioxidants.

### **2. Materials and methods**

#### **2.1 Materials**

Raw milk from French-Alpine goats, raised at the International Goat Research Center, Prairie View A&M University, Prairie View, Texas, USA, was obtained. Raw milk with a fat content of 4.1% (wt/wt) that was determined according to the

**115**

**Table 1.**

*mutabilis seed oil (NHMO).*

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

method of Kleyn et al. [21] was collected during the early lactation period. Iron and copper contents in raw goat milk were determined by atomic absorption spectrometry using a Varian SpectrAA 55 (Varian Analytical Instruments, Inc., Walnut Creek, CA, USA). Raw goat milk was dry-ashed in a muffle furnace (Barnstead/ Thermolyne Corp., Dubuque, IA, USA) at 550°C. Ashes were dissolved in 0.2% nitric acid solution. The concentrations of iron and copper in raw goat milk were determined from the calibration curves that were produced under the same experi-

Algae oil was provided by Nutrinova Inc. (Somerset, NJ, USA). Algae oil was subjected to chromatography to remove peroxides, carotenoids, tocopherols, and other antioxidants, as previously described [22]. The chromatographically purified algae oil has a DHA concentration of 42.9% (**Table 1**). The fatty acid composition of chromatographically purified algae oil was determined by preparation of methyl esters [23], which were analyzed by gas chromatography–mass spectrometry (GC–MS). For the fatty acid profile of raw goat milk, the samples were centrifuged at 10,000× g for 1 h to harvest milk fat. Fatty acids of milk fat (% wt/wt) were directly methylated by *in situ* transesterification as described [24] and analyzed by GC-MS (Agilent model 7890A GC system attached to an Agilent model 5975C mass detector; Agilent Technologies Inc., Santa Clara, CA, USA) on a 30 m × 0.25 mm internal diameter, 0.25 μm film thickness capillary column. Methyl ester of 10,

*Chemical composition, peroxide value and anisidine value of goat milk (GM), chromatographically purified algae oil (PAO), chromatographically purified Hibiscus mutabilis seed oil (PHMO), and natural Hibiscus* 

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

mental conditions with known standards.

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

method of Kleyn et al. [21] was collected during the early lactation period. Iron and copper contents in raw goat milk were determined by atomic absorption spectrometry using a Varian SpectrAA 55 (Varian Analytical Instruments, Inc., Walnut Creek, CA, USA). Raw goat milk was dry-ashed in a muffle furnace (Barnstead/ Thermolyne Corp., Dubuque, IA, USA) at 550°C. Ashes were dissolved in 0.2% nitric acid solution. The concentrations of iron and copper in raw goat milk were determined from the calibration curves that were produced under the same experimental conditions with known standards.

Algae oil was provided by Nutrinova Inc. (Somerset, NJ, USA). Algae oil was subjected to chromatography to remove peroxides, carotenoids, tocopherols, and other antioxidants, as previously described [22]. The chromatographically purified algae oil has a DHA concentration of 42.9% (**Table 1**). The fatty acid composition of chromatographically purified algae oil was determined by preparation of methyl esters [23], which were analyzed by gas chromatography–mass spectrometry (GC–MS). For the fatty acid profile of raw goat milk, the samples were centrifuged at 10,000× g for 1 h to harvest milk fat. Fatty acids of milk fat (% wt/wt) were directly methylated by *in situ* transesterification as described [24] and analyzed by GC-MS (Agilent model 7890A GC system attached to an Agilent model 5975C mass detector; Agilent Technologies Inc., Santa Clara, CA, USA) on a 30 m × 0.25 mm internal diameter, 0.25 μm film thickness capillary column. Methyl ester of 10,


**Table 1.**

*Biochemistry and Health Benefits of Fatty Acids*

hydrogen to the lipid radical [16, 17].

The omega-3 fatty acids in the milk of grass-fed goats are predominantly linolenic acid (C18:3*n*-3), or alpha (α)-linolenic acid (ALA). DHA can be synthesized from dietary ALA, but the human body can only make very small amounts of DHA from ALA [9]. Therefore, there is a need to supplement foods with DHA. The addition of DHA from algae oil in food emulsions such as in goat milk emulsion requires the need for antioxidants. Antioxidants increase the shelf life of emulsions, but a clean label ingredient is required when added to milk. Oxidation in oil-in-water emulsions is thought to occur at the interface region between the oil and the aqueous phases [10]. In the oil phase of the emulsions, fatty acids are the target of free radicals i.e., hydroxyl radicals, which stimulate lipid peroxidation. Nonpolar antioxidants such as tocopherols and ascorbyl palmitate have been shown to be highly effective in protecting oil-in-water emulsions [11]. Tocopherols are free-radical terminators thereby, interrupting the free-radical chain of oxidative reactions by contributing hydrogen from the phenolic hydroxyl groups [12]. Ascorbyl palmitate, a lipid-soluble antioxidant, exhibits antioxidant activities that include single oxygen quenching and free-radical scavenging [13–15]. Ascorbyl palmitate has been shown to work synergistically with tocopherols by donating a hydrogen to the tocopheroxyl radical, formed as a result of tocopherol donating a

The oxidative stability of DHA-enriched emulsion may also be accomplished by the addition of vegetable oil to algae oil. In this context, one of the strategies developed to protect fish oil in a cow milk emulsion against oxidation was the mixing of rapeseed oil with fish oil prior to emulsification of cow milk [18]. The authors found that tocopherol isomers in concentrations similar to those found in natural rapeseed oil, and added to rapeseed oil stripped of natural tocopherols, significantly inhibited oxidation in cow milk emulsions enriched with fish oil [18]. *Hibiscus mutabilis* (Malvaceae) are shrubs with peach color flowers and originally native of China. The seeds of *Hibiscus mutabilis*, which do not have economic applications yet, are a source of vegetable oil. Although not widely reported in the literature, a high content of phenolic compounds, tocopherols are found in *Hibiscus mutabilis* seed oil. The seeds of *Hibiscus mutabilis* are also a source of lectin. Lectin from the seeds of *Hibiscus mutabilis* has carbohydrate-binding specificity to galactonic acid, which potently inhibited HIV-1 reverse transcriptase [19]. HIV, the RNA virus that causes AIDS, gradually disrupts the immune system in humans. Since a recent study suggested that DHA in high DHA-concentrated fish oil positively contributed to certain aspects of immune function in middle-aged obese adults [20], DHA-enriched goat milk stabilized by *Hibiscus mutabilis* seed oil potentially

can be used as immune stimulator for the adjunctive therapy of HIV.

In the present work the suitability of *Hibiscus mutabilis* seed oil for enhancing the oxidative stability of DHA-enriched goat milk emulsion was studied. Based on the potential synergistic effects of ascorbyl palmitate with tocopherols, it was assumed that the most efficient oxidative stabilization during homogenization and storage of DHA-enriched goat milk may be achieved by combining both of these

Raw milk from French-Alpine goats, raised at the International Goat Research Center, Prairie View A&M University, Prairie View, Texas, USA, was obtained. Raw milk with a fat content of 4.1% (wt/wt) that was determined according to the

**114**

lipophilic antioxidants.

**2.1 Materials**

**2. Materials and methods**

*Chemical composition, peroxide value and anisidine value of goat milk (GM), chromatographically purified algae oil (PAO), chromatographically purified Hibiscus mutabilis seed oil (PHMO), and natural Hibiscus mutabilis seed oil (NHMO).*

13-nonadecadienoate (Nu-Chek-Prep U-58M, Elysian, MN, USA) was used as an internal standard. The concentrations of tocopherols in the raw goat milk and the chromatographically purified algae oil were determined by reversed-phase highpressure liquid chromatography (HPLC) [25], and the results were expressed as μg/g of oil. The content of free fatty acids in the raw goat milk and the chromatographically purified algae oil was determined by AOAC method [26]. The fatty acid composition, the concentrations of tocopherols, the peroxide value (PV), the anisidine value (AV), and the content of free fatty acids in the raw goat milk and chromatographically purified algae oil samples are presented in **Table 1**.

The lipid-soluble antioxidant, ascorbyl palmitate, was purchased from DSM Nutritional Products, Inc. (Parsippany, NJ, USA). All reagents used were of analytical grade, ACS certified or HPLC grade, from Sigma-Aldrich (St. Louis, MS, USA). Deionized water was prepared by passing distilled water over a mixed bed of cation-anion exchanger and was used throughout this study.
