**7. Safety and efficacy of probiotics**

### **7.1 Safety considerations**

Probiotics formulated for use in mass-rearing facilities have been shown to be beneficial due to their ability to improve a multitude of parameters and contribute to the restoration of dysbiosis in the medfly digestive tract. The probiotics selected so far are exclusively from the family of Enterobacteriaceae, and they are the cause of enteric human diseases that can lead to illness and death [114]. The use of Enterobacteriaceae in medfly mass-rearing procedures is still under experimentation; researchers have not yet addressed the issue of handler safety and environmental risk in general. The use of the probiotic in the larval rearing medium at the rearing facility and the administration of the probiotic to the adult sterile males intended for release are the two processes to be considered for safety issues. In the first case, it has long been recognized that facility workers can become infected by the agents they manipulate, thus making the nature of their work an occupational hazard. In the second case, introducing pathogenic bacteria into the adult diet allows bacteria to be transmitted horizontally to the environment. Implementing biosecurity procedures in rearing units, such as daily decontamination of all surfaces and equipment with specific disinfectants and limiting ventilation inside production modules, is difficult and will incur additional costs. However, it is clear that an increasing number of experiments are based on the use of the inactivated form of the probiotic, which is prebiotic, which appears to be less complicated to handle and yields comparable results [29, 31, 35].

### **7.2 Microencapsulation of probiotics for medfly mass-rearing**

Acidulants are present in the mass-rearing medfly larval diet and play an important role in preventing microorganism growth, buffering diets, decreasing diet rancidity, and modifying the viscosity and consistency of the diet [115]. The pH of the larval diet is adjusted to 3.5–4.5 in insectaries. Acid stress inhibits bacterial proliferation and changes the phenotypes and morphology of bacterial cells in the medfly diet as a result [116, 117]. This is not in the probiotic's favor because it will be subjected to pre-ingestion stress, reducing its stability and effectiveness. Encapsulation will stabilize the probiotics during processing, storage, and the site of action to safeguard them in the medfly diets. Given that edible polymers can be used as coating materials to

provide a protective environment for the long-term viability of microorganisms, encapsulation is a successful food industry technique [118]. The polymer systems used to encapsulate probiotics are alginate, carrageenan, gelatin, chitosan, cellulose acetate phthalate, locust bean gum, modified starch, chitosan, gellan, xanthan, gum arabic, and animal proteins [119].

Probiotic encapsulation in mass-rearing is a new and unexplored area. Remarkably, some research has suggested that entomopathogenic bacteria be microencapsulated for pest control. Due to its low residual activity in the field, the most notable example is the microencapsulation of *Bacillus thuringiensis* (B.t.) with arabic gum, gelatin, and chitosan against some Coleoptera, Lepidoptera, and Hemiptera at larval and adult stages. Laboratory tests on *Trichoplusia ni* larvae (Lepidoptera: Noctuidae) revealed that the microencapsulation process had no effect on B. t. bioactivity. After 12 days, the mean number of larvae in microencapsulated formulations in colloidosomal microparticles (50 mm) was significantly lower than in a commercial B. t. formulation, and the effect of microencapsulated formulations was comparable to that of a chemical pesticide (lambda-cyhalothrin) [120]. The spray dryer produced a particle size of 32 nm against *Helicoverpa armigera* (Lepidoptera: Noctuidae) larvae damaging cotton, and the results show that even low doses of this encapsulation significantly reduced the larval population [121]. These and other experiments show promise for the use of microencapsulation to ensure the stability of probiotics throughout the medfly rearing process while paying attention to functionality, which is impaired in some experiments [122].
