**2.3 Oil extraction of** *Hibiscus mutabilis* **seeds**

The ground fractions of *Hibiscus mutabilis* seeds were placed in a filter paper (Whatman No. 42) and introduced in a cartridge and they were extracted in a Soxhlet extractor (Southern Labware, Inc., Cumming, GA, USA) using hexane at 65–70°C during approximately 5 h, the time necessary to extract most of the oil from the seeds. The solvent was then evaporated by a vacuum dryer (Columbia International Tech, Irmo, SC, USA), and the oil yield was 9.0 g from 100 g of seeds. The extracted *Hibiscus mutabilis* seed oil was transferred into glass tubes, centrifuged at 12,000× g for 30 min at room temperature, and then stored at 4°C in the dark until analyses. This oil is referred to as the natural *Hibiscus mutabilis* seed oil (**Table 1**). The natural *Hibiscus mutabilis* seed oil was subjected to chromatography to remove naturally occurring antioxidants such as tocopherols and carotenoids [22]. Thus, this

**117**

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

*Hibiscus mutabilis* seed oil is void of antioxidants and peroxides. The percent fatty acid composition of the chromatographically purified *Hibiscus mutabilis* seed oil and the natural *Hibiscus mutabilis* seed oil were determined according to procedures described [23] by GC–MS. The fatty acid composition of the chromatographically purified *Hibiscus mutabilis* seed oil was similar to the natural *Hibiscus mutabilis* seed oil (**Table 1**). The content of free fatty acids in the chromatographically purified *Hibiscus mutabilis* seed oil and the natural *Hibiscus mutabilis* seed oil was determined by AOAC method [26]. The concentrations of tocopherols, the PV, the AV, and the concentrations of free fatty acids of the natural and the chromatographically puri-

Three liters of raw goat milk was pasteurized by heating at 72°C and holding milk at this temperature for 15 s. 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 ascorbyl palmitate (200 μg/g of oil) were added to goat milk. Goat milk samples were then cooled to 50°C and immediately homogenized at 22.5 MPa (3263.35 psi) through a highpressure TC5 homogenizer (Stansted Fluid Power, Harlow, UK). The goat milk emulsion samples were transferred to sterile 100-ml Pyrex dark brown glass bottles, which were flushed with nitrogen and then stored at 2°C in the dark for 14 days. The goat milk emulsions, with added oils, were labeled as follows: PAO = chromatographically purified algae oil, PHMO = chromatographically purified *Hibiscus mutabilis* seed oil, NHMO = natural *Hibiscus mutabilis* seed oil, and LAAP = lipid-

The particle size of the oil droplets in the goat milk emulsions was measured at day 1 and day 14 at 21 ± 1°C with a SALD-2101 laser diffraction particle size analyzer (Shimadzu Corporation, Columbia, MD, USA). The emulsion samples were diluted 100 times with double deionized water before they were transferred into the chamber of the instrument. Particle size measurements in μm were carried out in

Lipids from the DHA-enriched goat milk emulsions were extracted by chloroform:methanol (1:1 wt/wt), using a small volume of solvent [27, 28]. The PV was measured directly on the oils or fats extracted from the DHA-enriched goat milk emulsions by colorimetric determination of iron thiocyanate [29]. This method measures primary oxidation products of oils or fats i.e., hydroperoxides of oils and fats. The mean measurements in meq/kg of three replicates were

The para (*p*)-anisidine value was determined in the DHA-enriched goat milk emulsions by AOAC method [30]. This method determines the amount of aldehydes (principally 2-alkenals and 2,4-dienals) present in the emulsion samples. The mean

fied *Hibiscus mutabilis* seed oil samples are presented in **Table 1**.

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

**2.4 Preparation of emulsions**

soluble antioxidant ascorbyl palmitate.

**2.6 Measurement of peroxide value**

**2.7 Measurement of p-anisidine value**

measurements of three replicates were reported.

**2.5 Droplet size measurement**

triplicate.

reported.

**Figure 1.** *Image of seeds of Hibiscus mutabilis.*

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

*Hibiscus mutabilis* seed oil is void of antioxidants and peroxides. The percent fatty acid composition of the chromatographically purified *Hibiscus mutabilis* seed oil and the natural *Hibiscus mutabilis* seed oil were determined according to procedures described [23] by GC–MS. The fatty acid composition of the chromatographically purified *Hibiscus mutabilis* seed oil was similar to the natural *Hibiscus mutabilis* seed oil (**Table 1**). The content of free fatty acids in the chromatographically purified *Hibiscus mutabilis* seed oil and the natural *Hibiscus mutabilis* seed oil was determined by AOAC method [26]. The concentrations of tocopherols, the PV, the AV, and the concentrations of free fatty acids of the natural and the chromatographically purified *Hibiscus mutabilis* seed oil samples are presented in **Table 1**.

## **2.4 Preparation of emulsions**

*Biochemistry and Health Benefits of Fatty Acids*

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

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

Fresh harvested seeds of *Hibiscus mutabilis* (**Figure 1**) were obtained from The Village Botanica, Inc. (Waller, TX, USA). The seeds were immediately frozen with liquid nitrogen until analysis. The frozen seeds were thawed, dried by air blower and then milled using a blender. The ground samples that passed through a 35-mesh

The ground fractions of *Hibiscus mutabilis* seeds were placed in a filter paper (Whatman No. 42) and introduced in a cartridge and they were extracted in a Soxhlet extractor (Southern Labware, Inc., Cumming, GA, USA) using hexane at 65–70°C during approximately 5 h, the time necessary to extract most of the oil from the seeds. The solvent was then evaporated by a vacuum dryer (Columbia International Tech, Irmo, SC, USA), and the oil yield was 9.0 g from 100 g of seeds. The extracted *Hibiscus mutabilis* seed oil was transferred into glass tubes, centrifuged at 12,000× g for 30 min at room temperature, and then stored at 4°C in the dark until analyses. This oil is referred to as the natural *Hibiscus mutabilis* seed oil (**Table 1**). The natural *Hibiscus mutabilis* seed oil was subjected to chromatography to remove naturally occurring antioxidants such as tocopherols and carotenoids [22]. Thus, this

chromatographically purified algae oil samples are presented in **Table 1**.

of cation-anion exchanger and was used throughout this study.

**2.2 Preparation of** *Hibiscus mutabilis* **seed samples**

**2.3 Oil extraction of** *Hibiscus mutabilis* **seeds**

sieve were used for oil extraction.

**116**

**Figure 1.**

*Image of seeds of Hibiscus mutabilis.*

Three liters of raw goat milk was pasteurized by heating at 72°C and holding milk at this temperature for 15 s. 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 ascorbyl palmitate (200 μg/g of oil) were added to goat milk. Goat milk samples were then cooled to 50°C and immediately homogenized at 22.5 MPa (3263.35 psi) through a highpressure TC5 homogenizer (Stansted Fluid Power, Harlow, UK). The goat milk emulsion samples were transferred to sterile 100-ml Pyrex dark brown glass bottles, which were flushed with nitrogen and then stored at 2°C in the dark for 14 days. The goat milk emulsions, with added oils, were labeled as follows: PAO = chromatographically purified algae oil, PHMO = chromatographically purified *Hibiscus mutabilis* seed oil, NHMO = natural *Hibiscus mutabilis* seed oil, and LAAP = lipidsoluble antioxidant ascorbyl palmitate.

#### **2.5 Droplet size measurement**

The particle size of the oil droplets in the goat milk emulsions was measured at day 1 and day 14 at 21 ± 1°C with a SALD-2101 laser diffraction particle size analyzer (Shimadzu Corporation, Columbia, MD, USA). The emulsion samples were diluted 100 times with double deionized water before they were transferred into the chamber of the instrument. Particle size measurements in μm were carried out in triplicate.

#### **2.6 Measurement of peroxide value**

Lipids from the DHA-enriched goat milk emulsions were extracted by chloroform:methanol (1:1 wt/wt), using a small volume of solvent [27, 28]. The PV was measured directly on the oils or fats extracted from the DHA-enriched goat milk emulsions by colorimetric determination of iron thiocyanate [29]. This method measures primary oxidation products of oils or fats i.e., hydroperoxides of oils and fats. The mean measurements in meq/kg of three replicates were reported.

#### **2.7 Measurement of p-anisidine value**

The para (*p*)-anisidine value was determined in the DHA-enriched goat milk emulsions by AOAC method [30]. This method determines the amount of aldehydes (principally 2-alkenals and 2,4-dienals) present in the emulsion samples. The mean measurements of three replicates were reported.

### **2.8 Statistical analysis**

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 determine significant differences among treatment means at p < 0.05.
