Future work and challenges

In recent years, the beneficial effects of lactoferrin containing probiotic products on health have been shown in clinical and animal studies (Tomita et al. 2009). However, the concept that the regulation of the intestinal microflora composition by probiotics and lactoferrin offers the possibility to influence the development of mucosal and systemic immunity, and prevention and treatment of some diseases, is still short of entire evidence. The human dietary intervention studies to investigate the hypothetical model shown in Fig. 6.1. appear very challenging.

6.2.1 Extended work based on the present study

The results in the present thesis have been supported by a recent study regarding the relative resistance of lactic acid bacteria to bovine lactoferrin and hydrolysed bovine lactoferrin (Chen et al. 2013). The special cell wall structures of probiotics or the protective materials related to their thick cell walls may contribute to this property. Further research may require observation of probiotic cell membranes using Atomic Force Microscopy (AFM) (Francius et al. 2008) and other appropriate technologies. On the other hand, the present study provided an indication that the selective antibacterial activity of L. reuteri DPC16 and its synergistic augmentation by bovine lactoferrin may have potential in the food industry for food preservation purposes. Much more work can be attempted in these applications.

Although the intestinal co-culture (Caco2/THP-1) model described in this thesis is complicated, the knowledge obtained from the initial attempts has provided useful information for future work. This model seems to be applicable and could reveal more insight into inflammatory bowel diseases. It may be a practical tool to bridge the gap between an in vitro study and an in vivo study in gut health research. The system is

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believed to be an ideal approach to reveal the mechanisms of inflammation related to intestinal barrier dysfunction, and possibly lead to the discovery of functional food solutions after further development and optimisation of the system. In addition, the discovery and use of stable DNA damage-inducers rather than the use of faecal water in general would be another goal to achieve a stable in vitro colon carcinogenesis model. Finally, in vivo studies are suggested to explore the safety and colonisation properties of

L. reuteri DPC16 in human subjects.

Figure 6.1 A hypothetical model of DPC16 + lactoferrin on intestinal disease prevention

6.2.2 Probiotic formulae in human GI intervention

Probiotic research is an interesting topic; studies in this field help understand the behaviour of GI microbiota in the human gut. However, it appears very challeging and

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too early to know how and why probiotics modulate or benefit the gut functions. The knowledge on human GI microbiota is not sufficient and it is still in the accumulating stage. Some new publications in the last three years start to answer the questions of “who is there’ and ‘what are they doing there”, and inadvertently arrive at the inquiry ‘how and why’ the GI microbiota functions (Korecka & Arulampalam 2012). Therefore, The major effects should be directed towards development of in vitro and in vivo

models to understand the human gut microbiota and explore development of biotherapeutic possibility and methods (Prakash et al. 2011).

The present study has indicated that viable cells of L. reuteri DPC16 in the lower intestine may be reduced significantly due to the transit through the GI harsh digestion conditions and also there is a debateable point as to whether administered L. reuteri

DPC16 cells are able to replicate and colonise in vivo. The composition of the human gut microbiota, as shown by the examination of faecal samples, has a remarkable stability (Savage 1977; Stebbings et al. 2002). Studies have shown that dietary supplementation is needed to introduce and maintain high levels of L. reuteri in hosts. Oral intake of L. reuteri has shown that faecal L. reuteri numbers increased rapidly within days of ingestion (Wolf et al. 1998). However, the question still remains as to whether its antimicrobial substances are able to suppress the growth of, and interfere with the adhesion of, pathogens in vivo. Similarly, with the other functions of antigenoxicity and immumodulation. Secondly, there is no doubt that the microbial profiles during the course of early childhood depend on the surrounding environmental exposure to both microbial and beneficial factors through breast milk (Palmer et al.

2007). Comparing to the stability of a core microbial community in the adult gut (Rajilic-Stojanovic et al. 2012), shaping the immature gut microbiota in infants could be less challenging. Thus, the outcomes of probiotic formulae intervention can be expected to be different in newborn and adult human models. Under the same principle, shaping the gut microbiota using a probiotic formula in a diseased human model could be different to that using a healthy human model. Hence, a carefully designed microencapsulated L. reuteri DPC16 and bovine lactoferrin combined formula is suggested to provide an opportunity to maintain higher bioavailability of both components and release within different timeframes to act synergistically in the gut. The microencapsulation studies should be extended to investigate not only how to maintain viable L. reuteri DPC16 cells in the target area but also how to keep them

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active and stay longer, ideally to develop live probiotic cell in situ “factories” that could deliver live bacteria or continually release antibacterial and bioactive substances to maintain a healthy colon. The human clinical trials should be properly designed to achieve long-term randomised controlled studies by selecting proper biomarkers for the changes of GI microbiota composition, GI metabolites and GI functions as the primary outcomes of gut health improvement and well-being. Notably, even when the results from randomised, placebo-controlled trials support the beneficial effects of a particular probiotic for a specific indication, the benefits are generally not translatable to other probiotic formulations (Fontana et al. 2013).