Alginate microcapsules as delivery and protective systems of Bacillus licheniformis in a simulated shrimp's digestive tract

Highlights

• Encapsulation of B. licheniformis in alginate microparticles was achieved through ionic gelation.

• Alginate microparticles protects B. licheniformis during in vitro digestion on shrimp's digestive juices.

• Alginate microparticles improve the amount of viable B. licheniformis in simulated shrimp's intestine.

Abstract

This work aims to encapsulate Bacillus licheniformis, a marine probiotic, in alginate microparticles (AMPs), and the evaluation of its controlled and targeted release within a simulated shrimp digestive tract (DT). The encapsulation process was carried out using the ionic gelation technique. Both free and bacteria-loaded AMPs were physicochemically characterized by size, morphology, surface electrical charge, the survival, and the number of encapsulated bacteria after the encapsulation process, and the bacterial survival after 40-days of storage (at 4 °C and 25 °C). The in vitro release and survival studies of the bacteria were carried out using a protocol developed in our laboratory by implementing buffers of dissected organs from shrimp's DT. Results indicated that microparticles with an average size of 172–185 μm and negatively charged (−16.77 and − 17.66 mV, respectively) were obtained after using the ionic gelation method. The bacterial survival and encapsulation efficiency showed high cell viability and yield above 99%. Stability studies showed that the best storage temperature was 4 °C, in which it remained almost 100% of the bacteria viable for 15 days; however, cell viability declined to 55% survival after 30 days of storage at this temperature. Regardless of the cell viability reduction after 30 days, there are enough viable bacteria cells to be considered as a probiotic product. Release and survival studies showed that alginate particles had a protective effect on bacteria by keeping ca. 51.29% of viable probiotic within the shrimp intestine; in contrast, free bacteria only reached the shrimp intestine ca. 27.16% viable. Our results suggest that microparticles can be produced by a low-cost method that could ultimately benefit shrimp farming in a near future.

Introduction

During the last decades, aquaculture has become one of the most dynamic industries worldwide, generating a significant economic impact, not only by food production but also by its contribution to livelihoods and income generation (Kumar and Engle, 2016). In Mexico, the farming of shrimp and other marine species represents an important activity that provides profits for producers, is a good source of food with high nutritional value, and gives a job opportunity for skilled and unskilled workers (Sosa-Villalobos et al., 2016). Diseases are the major threat that constrains the growth of different shrimp species in Mexico, generating thus, disastrous declines in production and significant economic losses for producers (Escobedo-Bonilla, 2016). For example, studies have reported that pathogenic bacteria (such as Vibrio sp., Aeromonas sp., Spirillum sp., and Flavobacterium sp.) are responsible for different affectations in shrimps, including necrotizing hepatopancreatitis, gill fouling, cuticle necrosis, and/or larval mortality (Escobedo-Bonilla, 2016; Noriega-Orozco et al., 2007). Therefore, new strategies that improve biosafety protocols are necessary today, to ensure an adequate control of these types of diseases that limit shrimp production (Lakshmi et al., 2013). The prevention and control strategies have been based mainly on the implementation of chemical additives and veterinary medicines, such as antibiotics (Chinabut and Puttinaowarat, 2005). Nevertheless, these preventive/control strategies may present significant risks to the health of diverse living organisms and to the aquatic environment, by promoting the propagation of resistant-bacterial strains, and by the accumulation of antibiotic residues on aquatic species (Holmström et al., 2003). The implementation of non-antibiotic agents, such as probiotics, has emerged as a promising strategy to enhance shrimp farming (Martínez Cruz et al., 2012). Probiotics are live microorganisms that promote health benefits on a host when are consumed in adequate amounts (Martínez Cruz et al., 2012). Also, probiotics have been related to positive effects on shrimp and other aquatic species by acting as growth promoters (Gobi et al., 2018), enhancing immune responses (Duan et al., 2018), and improving the water quality by modifying the presence of other microorganisms in water and soil (Yuvaraj and Karthik, 2015). Swapna et al (2015) studied the effect of the combination of two different probiotic bacteria, B. licheniformis, and Lactobacillus rhamnosus, on the growth of pacific white shrimp (Litopenaeus vannamei); results showed a synergic effect of both bacteria by increasing different growth indices (specific growth rate, average body weight, net weight gain, and average daily weight gain) of white shrimp in a culture pond environment. So, it seems that B. licheniformis could have a promising role within sustainable aquaculture. Recently, our group isolated a B. licheniformis strain (BCR 4–3) from guts of Crassostrea gigas, C. corteziensis, and Atrina maura; results showed that isolated strains presented basic characteristics (such as high tolerance to pH and salinity, high adhesion potential, high autoaggregation, biofilm formation, and resistance to antibiotics) to be used as probiotic in mollusk larvae and crustaceans cultures (Escamilla-Montes et al., 2015). This bacteria strain was used as a preventive agent for shrimp culture established in the area of Sinaloa, Mexico; however, results were not favorable, because the number of viable bacteria that reached and colonized the DT of shrimps was not enough to generate the desired beneficial effect. For this reason, we aimed to improve the use of B. licheniformis by its encapsulation in maltodextrin microparticles developed by spray-drying technology. Again, the results were not promising since the amount of viable bacteria in microparticles after the drying process, was not enough to be considered as a probiotic product.

Therefore, the present work aims to encapsulate B. licheniformis in sodium AMPs created by the ionic gelation method. We also study the impact of the encapsulation process on the targeted release of probiotic bacteria in a simulated DT of shrimp. Our methodology could be used as the basis to determine the ideal encapsulation and protection conditions of different probiotic bacteria with potential application as preventive or control agents in shrimp farming.