**4. Preparation of alginate gel-forming beads**

Beads can be prepared by gelation method. This method, it has undergone changes that are mentioned below:


#### **4.1 Syringe or dropper method**

This method includes the formation of hydrogel beads by alginate solution with a solution containing multivalent ions (mainly with two positive charges). Multivalent ions help the formation of willows by forming a bond with alginate. This is the most common method used and can be modified in many ways to provide the desired shape, size, and therapeutic effects. Sodium alginate solution is prepared in different concentrations. The drug (if it needs a suitable carrier) is added to this solution, the said mixture is stirred and enough time is provided for it to stabilize. This mixture is then added drop by drop to the solution containing divalent ions (different concentration) in different pH conditions. As a result, the formed willows are washed with different mixtures and dried under different conditions. Wet willows can be coated with polymers such as chitosan and then dried to change the release. Sodium alginate solution is mixed with drug solution and polymer, such as chitosan is added to this mixture. Then the resulting mixture is gelled with a solution containing divalent ions in the above form. More changes can be applied by adding alginate solution to the mixture of divalent ion salt and polymer (chitosan). Then the formed willows can be gelled through similar processes.

### **4.2 Extrusion method**

Extrusion is the process of making something with specific dimensions by pushing or pulling material through a die with a desired cross-section. The sodium alginate solution is mixed with the drug solution and transferred drop by drop into the twovolume metal salt solution by extrusion through a silica tube using a peristaltic pump. This method can be used for all the above-mentioned samples of medicine, alginate solution, and multi-capacitance electrolyte solution of gels. The resulting willows are filtered and dried at the optimum temperature.

#### **4.3 Laminar jet break-up or prilling method**

This method is a process of preparing small droplets through an upward airflow when in contact with a sodium alginate solution at a constant temperature and moving downward. To make willows, a vibrating nozzle tool is pumped into the solution of divalent metal salts at different speeds through the nozzle of solution drops. Finally, the formed willows are filtered and dried at the optimum temperature. This method has been widely used to produce microparticles with a very small dimensional range and high-encapsulation efficiency, especially in biotechnology for cell stabilization.

As a result, it can be said that according to different needs in the drug delivery industry, alginate polymer, which is purified from brown algae, can be widely used in modern drug delivery due to its various advantages, including biocompatibility [27, 28].

## **5. Modification of alginate by processing methods**

As the properties of polysaccharides are tightly related to their structures, the modification of alginate sometimes affects its properties. The molecular weight reduction of the alginate occurs through a uronic acids release by proton-catalyzed hydrolysis in acid conditions (pH < 5), and elimination reaction in neutral and alkaline conditions (pH > 5) [29, 30]. Microwave-assisted acidic hydrolysis of alginate has the same effect as normal acid hydrolysis, but it accelerates the reaction [31]. Ultrasound treatments of alginate at different frequencies cause polymer structure degradation, rearrangement, and alteration of its molecular weight, reducing the M/G ratio (changing hydrophobic interactions). As a result, they are harder than untreated polymers [32, 33]. High-power electrical energy (several tens of kilojoules) is used in the high-voltage electrical discharge method. If the electric field is strong enough, an electron avalanche will be the starting point for the spread of the streamer from the high-voltage needle electrode to the plate electrode. High-pressure shock waves, bubble cavitation, and fluid turbulence are produced and lead to partial decomposition and damage to the cell wall, which accelerates the extraction of biomolecules from biomass. Studies have shown that the molecular weight of alginate extracted by applying the high-voltage method was similar to that extracted with the classical method but had a higher polydispersity. Alginate fragmentations and degradations occurred leading to heterogeneity in Mw distribution. This method had logically no effect on the sequence ratio of this biopolymer [34]. Molar mass, polydispersity, and the intrinsic viscosity of alginate fall simultaneously during ultra-high-pressure homogenization (HPH) without any change in its conformational structure [35].

## **6. Applications of alginate**

Alginate is extracted from seaweed composed of a linear copolymer of D-mannuronic and L-guluronic acid monomers. These films are demonstrated to be effective against *L. monocytogenes*, *E. coli*, and *Salmonella typhimurium* [36]. Alginate is widely used in many fields such as cell immobilization, tissue engineering, microencapsulation of detergents and pharmaceuticals, as well as coating and edible film for food products [29, 37].
