Probiotics and prebiotics for human health: Innovation and new trends. Design of new functional foods and medical devices with pro- and prebiotic to improve intestinal and vaginal health

The intestinal microflora can be considered as a postnatally aquired organ composed of a large diversity of bacterial cells that can perform different functions for the host. This organ is highly exposed to environmental influences and thus odulated in its composition and functions by external factors, such as nutrition. Specific components of the intestinal microflora, including lactobacilli and bifidobacteria, have been associated with beneficial effects on the host, such as promotion of gut maturation and integrity, antagonisms against pathogens and immune modulation. The antimicrobial and antifungal activities of two probiotic strains, Lactobacillus rhamnosus IMC 501® and Lactobacillus paracasei IMC 502®, and their 1:1 combination, named SYNBIO® were studied using four different methods. Using two modified streak methods and a well diffusion method, the inhibitory activity of the probiotics and their metabolites towards six Gram+, nine Gram- pathogenic bacterial strains and eight Candida strains, was tested. Antagonistic effect of probiotic Lactobacillus strains was also investigated by co-culturing assay highlighting a significant inhibition of most of the pathogens tested in this study. The combination SYNBIO® showed a microbicidal activity against most of the strains tested in the study. Compared to the control, most of the pathogenic bacteria and yeast were inhibited by all probiotic strains tested to various degrees. Screening Lactobacillus strains according to their activity in various environmental conditions could precede the clinical efficacy studies for adjunct treatment with probiotics in cure of different gastrointestinal and vaginal tract infections. There is also evidence suggesting that the consumption of fermented products containing lactobacilli and bifidobacteria may play a role in preventing cancer of the colon and rectum but the mechanisms responsible for this effect are not fully understood and may be linked to different factors. Colon cancer is a major cause of cancer death in affluent countries and diet plays an important role in its development. One of the essential component of the colonic cancer genesis is represented by DNA damage. Gut microbial products and food components are thought to be principally responsible for the damage that initiates disease progression. Two probiotic strains, L. rhamnosus IMC 501® and L. paracasei IMC 502®, and their combination, named SYNBIO® were incubated at three different bacterial concentrations with a genotoxic human faecal water. HT29 cells were used to investigate the protective effects of the probiotic bacteria on DNA damage faecal water-induced by single cell gel electrophoresis (Comet) assay. The probiotic strains tested were not genotoxic and significantly decreased DNA damage induced by faecal water, indicating the potential to inhibit initiation. Incubation of faecal water with different concentrations of probiotics revealed that the decrease in genotoxicity was dose dependent. In this study it was also investigated the influence of both probiotic strains on protein expression and MAPKs signalling pathways in HT29 cells, showing an improvement of the intestinal epithelium permeability. Consumers are more aware and concerned about their lifestyle than ever before. This has increased demand for foods that promote health and wellness, such as functional products containing probiotics and prebiotics, which have a beneficial effect on the balance of intestinal microbiota. Fermented dairy products are generally good food matrices for probiotics, but the consumption of these products is limited due to growing vegetarianism and the large number of individuals who are lactose intolerant or on cholesterol-restricted diets. Thus, the development of non-dairy probiotic products, including food matrices based on fruit, vegetables and cereals, has been widely studied. This paper reviews the main applications of probiotics in dairy and non-dairy food products and also in products of vegetable origin and the characteristics that enable the use of these food matrices as potential carriers of probiotic bacteria. However, in order to be beneficial, the bacterial cultures have to remain live and active at the time of consumption. One of the aim of this study was to develop new probiotic food products, such as seasoned cheeses, salami, chocolate and ice-cream with a final probiotic concentration of approximately 109 CFU/daily dose of L. rhamnosus IMC 501® and L. paracasei IMC 502® mixed 1:1 (SYNBIO®). The survival and viability of probiotics were determined during the foods shelf-life. The values of viable probiotic bacteria of all dairy and non-dairy foods were between 107 and 109 CFU/g of food at the end of the shelf-life and for some of them the values were maintained even after the expiry date. Based on the results of the current study, all the dairy (“Caciotta” cheese, “Pecorino” cheese, “Büscion” Swiss cheese and “Fiordilatte” ice-cream) and non-dairy (“Ciauscolo” salami, Larded salami, Swiss small salami, milk chocolate, dark chocolate, organic jam and chocolate mousse) food products studied would be excellent vehicles to deliver the probiotic health effects because of the high viability of probiotics during the shelf-life of foods and in some cases even after their expiry date. The most well known food-based strategies to modulate the composition of the intestinal microbiota are the dietary use of prebiotics, probiotics and their combination, synbiotics. Since fermented foods have a great significance because they provide and preserve large quantities of nutritious foods in a wide diversity of flavours, aromas and texture, which enrich the human diet, another aim of the study was to screen the effect of buckwheat flour and oat bran as prebiotics on the production of probiotic fibre-enriched fermented milks, by investigating the kinetics of acidification of buckwheat flour- and oat bran-supplemented milk fermented by L. rhamnosus IMC 501®, L. paracasei IMC 502® and their 1:1 combination named SYNBIO®. The probiotic strains viability, pH and sensory characteristics of the fermented fibre-enriched milk products, stored at 4°C for 28 days were also monitored. The results showed that supplementation of whole milk with the tested probiotic strains and the two vegetable substrates results in a significant faster lowering of the pH. Also, the stability of L. rhamnosus IMC 501®, L. paracasei IMC 502® and SYNBIO® during storage at 4°C for 28 days in buckwheat flour- and oat bran-supplemented samples was remarkable enhanced. The aim of this preliminary fermentation was to develop a new synbiotic product using the best combination of probiotics and prebiotics by promoting better growth and survival and be acceptable to the consumers with high concentration of probiotic strain. This new product was used to conduct a human feeding trial to validate the fermented milk as a carrier for transporting bacterial cells into the human gastrointestinal tract. The probiotic strains were recovered from faecal samples in 40 out of 40 volunteers fed for 4 weeks one portion per day of synbiotic fermented milk carrying about 109 viable cells. Functional foods such as probiotics and/or prebiotics have the potential to prevent and treat gastrointestinal and vaginal infections and diseases. Probiotics and/or prebiotics are an important field of further research. Further well-conducted clinical studies using validated outcome measures are recommended. Among others, issues to be addressed are to further identify populations that would benefit most from probiotics/prebiotics administration, to determine the most effective dosing schedules, and to address the cost effectiveness of using probiotics and/or prebiotics for preventing and treating gastrointestinal and urovaginal infections. Undoubtedly, probiotic bacteria are most effective when safely and adequately integrated into one’s diet. However, if used for therapeutic purposes, they should be ingested via capsules or tablets. It is important to remember that when used in pediatrics, as a preventive approach (e.g. in the case of acute diarrhea, antibiotic-induced diarrhea and allergy) their long-term use is more practical if the chosen bacteria are incorporated in the diet through yogurt, fermented milk, dairy and non-daity products or other beverages or foods consumed during the weaning process. Relative to daily supplement use, this approach helps with compliance and it reduces costs. The probiotic strains L. rhamnosus IMC 501® and L. paracasei IMC 502® are used as deliverers in capsules or powders rather than in food form. Probiotic integrators/supplements maintain for a long period of refrigerated and/or room temperature storage a high bacterial concentration, and can be sprinkled directly onto food; stirred into beverages, or taken as capsules. As the intestinal microbiota, also vaginal microbiota can change composition rapidly, for reasons that are not fully clear. This can lead to infection or to a state in which organisms with pathogenic potential coexist with other commensals. The most common urogenital infection in women is bacterial and fungal vaginosis. The majority of cases are caused by Candida albicans, but also C. glabrata, C. krusei and C. tropicalis can be problematic. In vitro studies have shown that Lactobacillus strains can disrupt bacterial vaginosis and yeast biofilms and inhibit the growth of urogenital pathogens. The use of probiotics to populate the vagina and prevent or treat infection has been considered for some time, but only quite recently have data emerged to show efficacy, including supplementation of antimicrobial treatment to improve cure rates and prevent recurrences. Therefore, we investigated the blockage of Candida pathogens adherence by Lactobacilli, under three possible mechanisms: exclusion by adhered Lactobacillus strains, displacement of adhered pathogens and competition for receptor sites (inhibition test). The inhibition results highlights a significant (P<0.05) competition of Lactobacillus plantarum 319, L. rhamnosus IMC 501®, L. paracasei IMC 502® and SYNBIO® against all the Candida strains. Since the SYNBIO® is a combination 1:1 of L. rhamnosus IMC 501® and L. paracasei IMC 502®, that are characterized as probiotics, it was used for the formulation of two new probiotic medical devices. Vaginal ovules and douches were produced using SYNBIO® as preliminary evaluation of different matrix and cell viability during 6 months. During the storage at room temperature for 6 months, Witepsol® was the matrix that showed the highest suitability to preserve viable microorganism thus increasing the shelf-life of the product respect to PEG formulation. The results suggest that the probiotic strains used in the present study could prevent colonization of the urogenital tract by relevant pathogens such as Candida strains through barrier and interference mechanisms (mainly exclusion and competition). An advantage for women is that they can self-administer the probiotics. Many more studies are needed to optimize the defensive properties of the vaginal microbiota, but the potential remains that the health of many women can be improved by probiotic intervention.