Article pubs.acs.org/Biomac
Biofilm-Like Lactobacillus rhamnosus Probiotics Encapsulated in Alginate and Carrageenan Microcapsules Exhibiting Enhanced Thermotolerance and Freeze-Drying Resistance Wean Sin Cheow and Kunn Hadinoto* School of Chemical and Biomedical Engineering, Nanyang Technological University, 637459 Singapore S Supporting Information *
ABSTRACT: Microcapsules containing high-density biofilmlike Lactobacillus rhamnosus probiotics, in place of planktonic cells, are developed in order to enhance the cell viability upon exposures to stresses commonly encountered during food lifecycle (i.e., heating, freeze-drying, refrigerated storage, and acid). The high-density (HD) capsules are prepared by in situ cultivation of the planktonic cells in the confined space of polysaccharide-based capsules (i.e., chitosan-coated alginate and carrageenan capsules). Compared to their planktonic counterparts, the HD capsules exhibit higher freeze-drying resistance (∼40×) and higher thermotolerance upon prolonged wet heat exposures at 60 and 70 °C (∼12−8000×), but not at higher temperatures even for short exposures (i.e., 80 and 100 °C). The enhanced viability of the HD capsules, however, is not observed during the refrigerated storage and exposure to the simulated gastric juice. The alginate capsules are superior to carrageenan owed to their better cell release profile in the simulated intestinal juice and storage viability. storage9,10 and also upon their incorporation into various food products, mostly dairy, such as milk, yogurt, and cheese.10−12 Moreover, depending on the probiotic strain, the microencapsulation has also been found to enhance the cell survival upon freeze-drying13 and mild heating at temperatures below 65 °C,14,15 which are often encountered during food processing. To date, research efforts on probiotic microencapsulation have focused mainly on the cell viability enhancement in the following three areas, i.e., (1) gastrointestinal fluid, (2) food product environments, and (3) refrigerated storage after freezedrying.16 One area that has been identified in numerous reviews16−18 to be lacking is the development of heat-resistant probiotic microcapsules that possess tolerance to elevated temperatures above 65 °C, where most free probiotic species will die.19 The development of such microcapsules would enable the incorporation of probiotics to nondairy products that require processing at temperatures above 65 °C (e.g., cereals, granolas, and chocolate bars), or those products that are exposed to high temperatures during their preparation for consumption (e.g., instant hot beverages and instant oatmeal). On this note, it is worth pointing out that an alternative to developing the heat-resistant probiotic capsules is to engineer heat-resistant probiotic strains via genetic modifications or stress response treatments.20−22 However, this approach is not only significantly more challenging and more costly but also the
1. INTRODUCTION A human adult harbors ten times more bacterial cells than human cells, most of which (>70%) are present in the gut;1 therefore, it is not surprising that modifications of the gut microbiota via consumptions of functional foods have significant health impacts. Consumption of probiotics, which are defined by the World Health Organization as “live microorganisms (bacteria or yeasts) which when administered in adequate amounts confer health benefits to the host,”2 represents the most straightforward method to modify the gut microbiota. The probiotic organisms confer their health benefits through inhibition of pathogen growths, maintenance of health promoting gut microflora, and stimulation of the host’s immune response.3 The health benefits associated with probiotic consumption range from alleviations of symptoms of lactose malabsorption and irritable bowel syndromes, to suppression of colon cancer and enhancing resistance to gut infections.4 To confer their health benefits, the probiotic cells must retain their viability during the three critical stages of (1) processing into food products, (2) storage, and (3) upon transit through the acidic stomach and intestine. In this regard, microencapsulation of probiotic cells into carrier matrixes, where polysaccharides (e.g., alginate, carrageenan, and chitosan) are typically used, has been found effective in protecting the cells from the acidic environment of the stomach and subsequently in facilitating the gradual cell release in the intestinal sections of the gut.5−8 In addition, in comparison to the free cells (i.e., nonencapsulated), the encapsulated probiotic cells in their freeze-dried form exhibit significantly higher viabilities during © 2013 American Chemical Society
Received: June 11, 2013 Revised: August 7, 2013 Published: August 16, 2013 3214
dx.doi.org/10.1021/bm400853d | Biomacromolecules 2013, 14, 3214−3222
Biomacromolecules
Article
Figure 1. Encapsulation of high-density biofilm-like probiotic cells, which are grown in situ from the encapsulated planktonic cells, is postulated to lead to enhanced stress resistance and thermotolerance.
Biofilm is a sessile high-density community of bacterial cells enclosed by a self-secreted extracellular polymeric substance (EPS) that forms a protective layer around the individual cells. Probiotics, including Lactobacillus and Bifidobacterium, have been found to naturally exist in the gut in their biofilm states adhered to the gut lining.28 Importantly, biofilm cells are widely known to exhibit greater resistance than the planktonic cells to antibacterial agents29 and various stresses present in their environment (e.g., acid and heat).30,31 The enhanced resistance in the biofilm has been attributed to (1) its dormant metabolic state and quorum sensing, both of which render biofilm cells innately robust, and (2) the shielding presence of the EPS.29 Recognizing the superior resistance of biofilm in general, we hypothesize that encapsulation of biofilm probiotic would also lead to higher cell survivals upon heating than the planktonic probiotic. To test the hypothesis, we prepare polysaccharidebased microcapsules containing biofilm-like probiotic cells by means of in situ cultivation of the planktonic cells inside the confined space of the capsule as illustrated in Figure 1, resulting in the growth of high-density colonies inside the capsules that are characteristics of biofilm. In addition to the thermotolerance, the capsules containing the biofilm-like probiotic cells are also examined in terms of their viabilities after (1) freezedrying, (2) refrigerated storage, and (3) exposure to simulated gastric juice. The results are then compared with the viabilities of the capsules containing the planktonic cells. We must emphasize that the objective of the present study is not to deliver ready-to-market heat-resistant probiotic microcapsules, but rather to serve as a proof-of-concept study in which we aim to demonstrate that encapsulation of the biofilmlike cells can result in higher cell viabilities upon exposures to stresses, which are commonly encountered in the preparation and consumption of food products. To this end, Lactobacillus rhamnosus GG is employed as the probiotic model owed to its clinically proven health promoting effects32 and its confirmed biofilm-forming ability in vitro. 33 The effects of the polysaccharide used as the capsule (i.e., alginate and carrageenan) on the stress resistance of the encapsulated cells and their release profile in simulated intestinal juice are investigated. Both microcapsules are coated with chitosan to ensure their gastrointestinal stability and also to function as a thermal protective layer.27 Alginate, carrageenan, and chitosan are chosen as they are commonly used for encapsulation of live bacterial cells owed to their biocompatibility and low cost.17
public acceptance of genetically modified food remains highly debated.23 Thus, encapsulation of probiotic cells into heatresistant capsules represents the more plausible and widely accepted approach. One of the very few studies on the heat-resistant probiotic microcapsules was by Chen et al.,24 who encapsulated Bifidobacterium bifidum in alginate-gellan gum microcapsules coated with peptides and prebiotics. Upon heating at 75 °C for 1 min, ∼90% of the encapsulated cells were shown to survive compared to 80% survival for the cells encapsulated in only alginate matrix and ∼70% survival for the free cells. Nevertheless, as B. bif idum is known to possess low thermotolerance (i.e.,
