**4. Extraction process**

Nowadays, there are numerous extraction methods of bioactive substances from nature for the application into functional food and pharmaceuticals. For example, maceration, reflux, soxhlet, microwave-assisted, ultrasonic-assisted, enzymeassisted, and gamma Coban 60-assisted. Different bioactive ingredients will have specifically suitable extraction methods, and the **Figure 4** will present the thing.

## **4.1 Collagen**

The high temperature leads to the degradation of collagen structure, so they are usually pre-treatment, extracted, and purified under cold temperatures (≤ 5°C). After pre-treatment, skins and scales will be dried by using infrared – assisted

#### **Figure 4.**

*General extraction schematic of antioxidant ingredients from organic materials.*

#### *Functional-Antioxidant Food DOI: http://dx.doi.org/10.5772/intechopen.96619*

cold drying and minced. For collagen extraction, the solvent of CH3COOH is usually useful (**Figures 5** and 6). For fish skins, removing non-protein and lipid are by using 0.1 M NaOH and 10% butyl alcohol, respectively (**Figure 5**). NaCl and ethanol are suitable for the precipitation of collagen from skins and scales, respectively.

Fish skin is scraped, washed to remove impurities, washed with cold water to remove impurities, then chopped with a size of 0.5 x 0.5 cm. After soaking for enough time, the fish skin is removed and rinsed with cold water until neutral pH. Fish skin will be extracted with 0.5 M CH3COOH solution at 1/15 (w/v) ratio for 48 hours and precipitated in 2.6 M NaCl in 0.05 M tris buffer (hydroxymethyl) aminomethane (pH 7.0) (**Figure 6**). The precipitate was then centrifuged at 20,000 g for 60 minutes at 4°C, then undergone dialysis and finally lyophilized to collect the collagen. The efficiency obtained from the bovine skin with acetic acid

**Figure 5.** *Extraction schematic of antioxidant collagen from fish scales.*

#### **Figure 6.**

*Extraction schematic of antioxidant collagen from fish skin.*

is 4.19% (calculated on the wet weight of the skin), the obtained collagen is types I that consisted of 2 chains α is α1 and α2, denaturation temperature is 31.16°C. According to the study, pangasius skin was treated with 0.2 M NaOH for 66 hours, extracted collagen in acetic acid 0.37 M for 2.5 days. Collagen precipitation occurs in 2.05 M NaCl in 4 minutes. The results showed collagen collection efficiency of 31.16%.

#### **4.2 Polysaccharide**

All over methods could use for polysaccharides extraction, but only three solvents could use, for example, alkaline, acid, and aqueous.

#### *4.2.1 Fucoidans*

For fucoidans extraction, removing the pigment and the lipid out of brown algae is by using an organic solvent such as ethanol/acetone/chloroform and HCl, respectively. Before the fucoidans extraction, separation of polyuronic acid is also via the other steps, such as neutralize (8% NaHCO3), concentration, dialysis (10 kDa membrane), and precipitation. Finally, fucoidans are collected with HCl solvent and running the DEAE cellulose (**Figure 7**).

*Functional-Antioxidant Food DOI: http://dx.doi.org/10.5772/intechopen.96619*

**Figure 7.**

*Extraction schematic of antioxidant fucoidan from brown algae.*

#### *4.2.2 Alginates*

The removal step of pigment and lipid by using an organic solvent and HCl could not be lack. Na2CO3 is a useful solvent for the extraction of alginate. The conversion of sodium alginate to calcium alginate is necessary. Calcium alginate still links to phlorotannins, so bleaching is important. Acidification (pH 2) is phlorotannins removal. Finally, the conversion of sodium alginate is from alginic acid, and the precipitation of them by using 80% ethanol (**Figure 8**) [70].

Based on experiments, the use of a hydrochloric solvent with flexibility in terms of temperature, the material-to-solvent ratio, and time, as well as neutralization, dialysis, concentration, precipitation, centrifugal, and drying, will allow obtaining small units of alginate such as sodium mannuronate and sodium guluronate (**Figure 9**).

#### *4.2.3 Glucosamine*

Shrimp shells contain many chitin, protein, and minerals, so removing protein and minerals using HCl and NaOH are indispensable steps. After removing proteins and minerals, continue to wash and soak chitin in HCl solvent to hydrolyze

**Figure 8.** *Extraction schematic of antioxidant alginate from brown algae.*

glucosamine. Glucosamine hydrochloride will be collected by crystallization with ethanol and dried at 50°C (**Figure 10**) [71].

#### *4.2.4 Inulins*

Extracting and purifying inulin from dangshen, color separation is also a necessary step from the beginning. After color separation, inulin is extracted with water and undergoes filtration, concentration, and purification using ethanol, activated carbon, and CaCl2. The above repetitions will be useful for purified inulin absorption (**Figure 11**).

*Functional-Antioxidant Food DOI: http://dx.doi.org/10.5772/intechopen.96619*

**Figure 9.** *Extraction schematic of antioxidant sodium mannuronate and sodium guluronate from alginate.*

#### *4.2.5 Laminarins*

As laminarin is a water-soluble polysaccharide sulfate, several proteins, fucoidan, and alginate also exist in the laminarin extract. Protein removal is using trichloroacetic acid. Eliminate alginate and fucoidan are by adjusting pH and mass fractionation, respectively (**Figure 12**) [72].
