Viability of probiotic Lactobacillus rhamnosus in structured emulsions containing saturated monoglycerides
Introduction
Modern consumers expect their food to be healthy and to prevent illness as they are increasingly interested in personal health (Bech-Larsen & Scholderer, 2007). Foods which promote health beyond providing basic nutrition are defined “functional foods”. This term covers a broad range of products that may contain one or a combination of components that have desirable cellular or physiological effects on the body. Functional foods can include probiotics, prebiotics, synbiotics, or bioactive compounds such as antioxidants, minerals, vitamins, active peptides (Champagne, Gardner, & Roy, 2005). Particularly, probiotic products are important functional foods representing about 65% of the world market of functional food and their demand is continuously growing (Agrawal, 2005). Food applications of probiotics are mainly in dairy products (such as yogurt, kefir, cultured drinks, cheese, ice cream, dairy desserts, infant formula), but also in other food categories, such as chocolate, cereals, juices, soya-based products (Champagne et al., 2005, Coman et al., 2012, Innocente et al., 2016).
According to FAO/WHO probiotics are defined as “live microorganisms which when administered in adequate amounts confer a health benefit on the host” (Araya et al., 2002). Today, the positive effects on human health of single or mixed microbial probiotic cultures have been widely demonstrated. However, to confer their beneficial effects to the host’s health, the viability of probiotic cells in food is of paramount importance. Populations of 106 to 107 CFU/g in the final product have been established as efficient to guarantee the health benefits of the probiotic cultures (Talwalkar, Miller, Kailasapathy, & Nguyen, 2004). This amount is equivalent to 108 to 109 CFU provided by a daily consumption of 100 g or 100 mL of the selected food (Jayamanne & Adams, 2006). Thus, to define a product as functional it is fundamental that probiotic bacteria remain metabolically active and viable after processing and throughout product shelf-life (Krasaekoopt, Bhandari, & Deeth, 2003).
Several factors could affect the viability of probiotics in foods, including low pH, hydrogen peroxide and dissolved oxygen content, presence of competing microorganisms and inhibitors, water activity, and processing and storage temperature (Coman et al., 2012, Kailasapathy, 2002). Different approaches aimed to increase the resistance of sensitive probiotics against adverse conditions have been proposed. Examples are the use of oxygen-impermeable containers, stress adaptation during cultivation, as well as the incorporation of micronutrients such as peptides and amino acids (Anal, 2007). Beside these strategies, a current trend is to design physical barrier surrounding probiotics able to protect them against adverse environmental conditions (Burgain et al., 2011, Kailasapathy, 2002). Immobilization and encapsulation are powerful technologies that could improve the protection level of probiotic cells during food processing and storage (Borgogna, Bellich, Zorzin, Lapasin, & Cesàro, 2010). It is a matter of fact that strategies allowing the protection of bacteria into foods could improve also their delivery in the human gut.
The terms immobilization and encapsulation were used interchangeably in most reported literature. While encapsulation is the process of forming a continuous coating around the probiotic cells that are embedded within the capsule wall as a core of encapsulated material, immobilization refers to the trapping of bacteria cells within or throughout a matrix (Kailasapathy, 2002). Clearly, the materials used as encapsulation/immobilization systems should be non-toxic and biocompatible. Researches were mainly focussed on the use of polysaccharides, such alginate (Chávarri et al., 2010, Cook et al., 2012, Mokarram et al., 2009), xanthan gum and starch (Burgain et al., 2011, Ding and Shah, 2009), and proteins, such as casein (Heidebach et al., 2009, Heidebach et al., 2010), whey protein (Doherty et al., 2010, Doherty et al., 2012) and gelatin (Borza et al., 2010). On the contrary, little and fragmentary information are available on the use of lipid matrices as encapsulating materials (Gomes-Da-Silva et al., 2012, Pedroso et al., 2012, Wolfe et al., 2014). Experimental data suggest that, when bacteria are encapsulated into solid fats, they are better protected against environmental stresses probably due to the presence of fat crystal network. For instance, Bifidobacterium longum encapsulated in cocoa butter showed higher viability than in starch (Lahtinen, Ouwehand, Salminen, Forssell, & Myllärinen, 2007) and Bifidobacterium animalis was more resistant to acid stress when encapsulated into a blend of crystalline hydrogenated palm stearin and palm kernel oil (Wolfe et al., 2014).
Based on these findings, the present research aimed to investigate the viability of a Lactobacillus rhamnosus probiotic strain included in emulsions structured by saturated monoglycerides. Saturated monoglycerides were selected due to their peculiar structuring capacity in multiphase environments. As extensively reported in literature, long chain saturated MGs self-assemble into highly hydrated lamellar phase in the presence of water above the critical Krafft temperature (Rogers, 2016). The lamellar phase is made of double layers of lipid molecules separated by water layers. When the MG-water mixture is cooled below the Krafft temperature, the so called α-gel phase appears. The peculiarity of these systems is that MG lamellar bilayers can create a shell entrapping a high quantity of oils resulting in gel like systems (Batte et al., 2007b, Valoppi et al., 2015a, Valoppi et al., 2015b). Thus, MG structures divide the hydrophilic and lipophilic domains by creating a compartmentalization that could be exploited to introduce guest molecules of hydrophilic, lipophilic or amphiphilic nature. Hydrophilic molecules could locate close to the emulsifier polar head or in the water domains, lipophilic molecules within the lipophilic domains and, finally, amphiphilic molecules at the interface (Sagalowicz, Leser, Watzke, & Michel, 2006). For instance, these structures have been adopted as aroma compounds delivery systems (Mao, Calligaris, Barba, & Miao, 2014) as well as carriers of omega-3 fatty acids in cheese and bakery product formulations (Anese et al., 2016, Calligaris et al., 2015). In our knowledge, no information is available in literature on microbial cell viability inside ternary systems composed of MG, an aqueous phase and a lipid phase. To understand the effect of the composition of ternary system, the viability of a commercial L. rhamnosus strain was studied into MG-structured emulsions differing in both aqueous and lipid phase composition. Three different aqueous phases were selected: an aqueous solution containing 1 mM potassium bicarbonate (KHCO3) or potassium carbonate (K2CO3) and UHT skim milk. The ability of saturated MG to structure these ternary systems was previously demonstrated (Valoppi et al., 2015a, Valoppi et al., 2015b). Beside different water phases, sunflower oil or anhydrous milk fats were considered as lipid phase to highlight a possible role of the physical state of the lipid matrix (liquid vs crystalline) included in the MG structures on bacteria viability. The viability of probiotic cells into the six considered structured emulsions was then tested just after preparation and during storage at 4 °C for up to 56 days.
Section snippets
Culture preparation
A commercial probiotic L. rhamnosus culture (Lyofast LRB) was purchased as a lyophilized pellet (Sacco s.r.l, Cadorago, Como, Italy). The lyophilized culture was propagated in MRS broth (Oxoid, Milan, Italy) and stored as a 20% (v:v) glycerol stock-culture at −80 °C. Overnight cultures of L. rhamnosus were prepared by subculturing stock-cultures in MRS broth (1% v:v inoculation) at 37 °C for 24 h under anaerobic incubation. L. rhamnosus was added to MG-based emulsions as lyophilized culture or as
Characterization of MG-ternary mixtures
Fig. 1 shows visual appearance and microstructure of MG-structured emulsions containing sunflower oil (a) or AMF (b) as lipid phase, respectively. In both cases, the water phase was made of an aqueous solution of KHCO3, K2CO3 or defatted UHT milk. All samples demonstrated a solid-like behaviour resulting in white systems. Observing microscopic images of samples containing oil, it can be noted that they were crowed of small oil droplets (dark zones) surrounded by crystalline monoglyceride shells
Conclusions
Results obtained highlighted the capacity of MG structures to ensure a high cell viability of L. rhamnosus. While the production of MG-structured emulsions is technologically feasible by using different aqueous and lipid phases, their use as vehicles for the delivery of probiotic cells would be successful only if the emulsions contain in the aqueous phase sufficient nutrients to support their metabolism. In this study, defatted milk appeared a good solution. On the contrary, the physical state
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