# Microencapsulation of Natural Food Antimicrobials: Methods and Applications
## 2 Encapsulation Methods
### Coacervation  
Coacervation (simple or complex) is the phase separation of one or many hydrocolloids from the initial solution with prepared coacervates as a wall and suspended or emulsified active agents as the core. The formation of complex coacervates is influenced by various parameters, such as pH, polymer concentration, the polymer mixing ratio and ionic strength. Appropriate enzymatic or chemical crosslinking agents can be applied for changing the liquid coacervate layer into a solid shell. The main advantages of encapsulation by coacervation are the high EE (up to 99%) and the excellent controlled-release properties [46]. One of the main challenges relating to coacervation is the addition of chemical crosslink such as glutaraldehyde and formaldehyde considered as highly toxic agents [47] but replacement of these agents with nontoxic crosslinkers, such as transglutaminase enzyme, tannic acid [48] and genipin extracted from _Gardenia [PERSON]_ plant [22], has been reported. In addition, some protein-polysaccharide systems were investigated without adding any crosslinker agents [23; 49; 50].  
Simple coacervation methods formed by a single hydrocolloid have been used for microencapsulation of antimicrobial compounds because of the inexpensive and flexible process and the easy formation of microcapsules containing hydrophobic substances such as holy basil and lemongrass (_Cymbopogon citrates_) EO [20; 21]. Regardless of the coacervation type, gelatin, an amphoteric protein consisting of carboxylic and amino guanidine groups, is considered as the most commonly used encapsulating material [21; 47].The production of microcapsules via complex coacervation is related to physical interactions between oppositely charged proteins (protonated amino groups) and polysaccharides (de-protonated carboxyl groups). The gelatin-gum arabic system has been mostly used for complex coacervation microencapsulation of flavors and EOs [51; 52; 53; 54]. The combination of wall materials and drying processes such as spray drying and freeze drying for producing microcapsules by complex coacervation has significant effects on the EE. However, degradation of active agents during encapsulation by spray drying can occur due to the use high temperatures (70-150 \({}^{\circ}\)C) applied in this process. Using lyophilization as a low-temperature method can lead to higher stability of heat-labile active compounds during the encapsulation process. However, the main disadvantages of lyophilization are that it is expensive and time-consuming [55].  
Amongst studied crosslinkers, genipin, a natural water-soluble crosslinker, has the advantage of acting rapidly in acidic pH compared to aldehyde substances that must be applied in basic conditions [38]. Mustard seed EO has been microencapsulated in gelatin-gum arabic complex coacervates with genipin as a crosslinker. Optimum crosslinking and complex coacervation processes occurred at 8 h, pH 10.0 and with 0.075 g of genipin per g of gelatin. Relative humidity and temperature are two important factors affecting storage stability of EO. Microencapsulated EOs showed a very slow release rate at a high temperature and in an humid environment [22]. Different protein-polysaccharide wall materials have been tested for the microencapsulation of active agents such as citronella essential oil, palm oil, garlic extract and cinnamon extract by complex coacervation [24; 56; 57; 58]). For instance, whey protein isolate (WPI) and chitosan with three deacetylation degrees (83%, 94% and 96%) were used for the encapsulation of garlic extract by complex coacervation followed by spray drying. High retention of phenolic compounds (61.40%) and better antioxidant properties were consequently obtained for chitosan-96%/WPI powder as a result of the excellent physicochemical characteristics related to the higher number of free amines interacting with protein. Being water-soluble, the produced microparticles can be applied in different food matrices.