Tiina Mattila-Sandholm

Probiotic bacteria: safety, functional and technological properties

During the past two decades probiotic (health promoting) micro-organisms have been increasingly included in various types of food products, especially in fermented milks. Several aspects, including safety, functional and technological characteristics, have to be taken into consideration in the selection process of probiotic micro-organisms. Safety aspects include specifications such as origin (healthy human GI-tract), non-pathogenicity and antibiotic resistance characteristics. Functional aspects include viability and persistence in the GI-tract, immunomodulation, antagonistic and antimutagenic properties. Before probiotic strains, chosen on the basis of their good safety and functional characteristics, can benefit the consumer, they must first be able to be manufactured under industrial conditions. Furthermore, they have to survive and retain their functionality during storage, and also in the foods into which they are incorporated without producing off-flavours. Factors related to the technological and sensory aspects of probiotic food production are of utmost importance since only by satisfying the demands of the consumer can the food industry succeed in promoting the consumption of functional probiotic products in the future.

Demonstration of safety of probiotics: a review.

Probiotics are commonly defined as viable microorganisms (bacteria or yeasts) that exhibit a beneficial effect on the health of the host when they are ingested. They are used in foods, especially in fermented dairy products, but also in pharmaceutical preparations. The development of new probiotic strains aims at more active beneficial organisms. In the case of novel microorganisms and modified organisms the question of their safety and the risk to benefit ratio have to be assessed. Lactic acid bacteria (LAB) in foods have a long history of safe use. Members of the genera Lactococcus and Lactobacillus are most commonly given generally-recognised-as-safe (GRAS) status whilst members of the genera Streptococcus and Enterococcus and some other genera of LAB contain some opportunistic pathogens. Lactic acid bacteria are intrinsically resistant to many antibiotics. In many cases resistances are not, however, transmissible, and the species are also sensitive to many clinically used antibiotics even in the case of a lactic acid bacteria- associated opportunistic infection. Therefore no particular safety concern is associated with intrinsic type of resistance. Plasmid-associated antibiotic resistance, which occasionally occurs, is another matter because of the possibility of the resistance spreading to other, more harmful species and genera. The transmissible enterococcal resistance against glycopeptide antibiotics (vancomycin and teicoplanin) is particularly noteworthy, as vancomycin is one of the last effective antibiotics left in the treatment of certain multidrug-resistant pathogens. New species and more specific strains of probiotic bacteria are constantly identified. Prior to incorporating new strains into products their efficacy should be carefully assessed, and a case by case evaluation as to whether they share the safety status of traditional food-grade organisms should be made. The current documentation of adverse effects in the literature is reviewed. Future recommendations for the safety of already existing and new probiotics will be given.

Lactic Acid Permeabilizes Gram-Negative Bacteria by Disrupting the Outer Membrane

ABSTRACT The effect of lactic acid on the outer membrane permeability ofEscherichia coli O157:H7, Pseudomonas aeruginosa, and Salmonella enterica serovar Typhimurium was studied utilizing a fluorescent-probe uptake assay and sensitization to bacteriolysis. For control purposes, similar assays were performed with EDTA (a permeabilizer acting by chelation) and with hydrochloric acid, the latter at pH values corresponding to those yielded by lactic acid, and also in the presence of KCN. Already 5 mM (pH 4.0) lactic acid caused prominent permeabilization in each species, the effect in the fluorescence assay being stronger than that of EDTA or HCl. Similar results were obtained in the presence of KCN, except for P. aeruginosa, for which an increase in the effect of HCl was observed in the presence of KCN. The permeabilization by lactic and hydrochloric acid was partly abolished by MgCl2. Lactic acid sensitized E. coli and serovar Typhimurium to the lytic action of sodium dodecyl sulfate (SDS) more efficiently than did HCl, whereas both acids sensitized P. aeruginosa to SDS and to Triton X-100. P. aeruginosawas effectively sensitized to lysozyme by lactic acid and by HCl. Considerable proportions of lipopolysaccharide were liberated from serovar Typhimurium by these acids; analysis of liberated material by electrophoresis and by fatty acid analysis showed that lactic acid was more active than EDTA or HCl in liberating lipopolysaccharide from the outer membrane. Thus, lactic acid, in addition to its antimicrobial property due to the lowering of the pH, also functions as a permeabilizer of the gram-negative bacterial outer membrane and may act as a potentiator of the effects of other antimicrobial substances.

Technological challenges for future probiotic foods

Abstract Modern consumers are increasingly interested in their personal health, and expect the food that they eat to be healthy or even capable of preventing illness. Gut health in general has shown to be the key sector for functional foods in Europe. The probiotic yoghurt market is well established but the key growth sector recently has been the probiotic drinks. The popularity of dose-delivery systems for probiotic drinks has also resulted in research efforts targeted to developing probiotic foods outside the dairy sector. New product categories, and thus novel and more difficult raw materials with regard to technology of probiotics, will certainly be the key research and development area for future functional food markets. The viability and stability of probiotics has been both a marketing and technological challenge for industrial producers. Probiotic foods should contain specific probiotic strains and maintain a suitable level of viable cells during the products shelf life. Unless strict demands are set on probiotic product definition and labelling their regulatory definition will remain obscure. The technological demands placed on probiotic strains are great and new manufacturing process and formulation technologies may often be required for bacteria primarily selected for their functional health properties. Before probiotic strains can be delivered to consumers, they must first be able to be manufactured under industrial conditions, and then survive and retain their functionality during storage as frozen or freeze-dried cultures, and also in the food products into which they are finally formulated. The probiotic strains should also survive the gastrointestinal stress factors and maintain their functionality within the host. Additionally, they must be able to be incorporated into foods without producing off-flavours or textures—they should be viable but not growing. The packaging materials used and the conditions under which the products are stored are also important for the quality of products. Future technological prospects exist in innovations finding solutions for the stability and viability problems of probiotics in new food environments. Current research on novel probiotic formulations and microencapsulation technologies exploiting biological carrier and barrier materials and systems for enteric release provides promising results. Maintenance of low production costs will remain the challenge for future probiotic process and formulation technologies. Exploitation of food-grade raw materials such as native, and physically or enzymatically treated starches, is one example of future technology that has the potential to meet the challenge of broadening the range of food types into which probiotic ingredients can be successfully incorporated. Novel developments for control release systems in foods and pharmaceuticals will also provide new possibilities.

Comparative genomic analysis of Lactobacillus rhamnosus GG reveals pili containing a human- mucus binding protein

To unravel the biological function of the widely used probiotic bacterium Lactobacillus rhamnosus GG, we compared its 3.0-Mbp genome sequence with the similarly sized genome of L. rhamnosus LC705, an adjunct starter culture exhibiting reduced binding to mucus. Both genomes demonstrated high sequence identity and synteny. However, for both strains, genomic islands, 5 in GG and 4 in LC705, punctuated the colinearity. A significant number of strain-specific genes were predicted in these islands (80 in GG and 72 in LC705). The GG-specific islands included genes coding for bacteriophage components, sugar metabolism and transport, and exopolysaccharide biosynthesis. One island only found in L. rhamnosus GG contained genes for 3 secreted LPXTG-like pilins (spaCBA) and a pilin-dedicated sortase. Using anti-SpaC antibodies, the physical presence of cell wall-bound pili was confirmed by immunoblotting. Immunogold electron microscopy showed that the SpaC pilin is located at the pilus tip but also sporadically throughout the structure. Moreover, the adherence of strain GG to human intestinal mucus was blocked by SpaC antiserum and abolished in a mutant carrying an inactivated spaC gene. Similarly, binding to mucus was demonstrated for the purified SpaC protein. We conclude that the presence of SpaC is essential for the mucus interaction of L. rhamnosus GG and likely explains its ability to persist in the human intestinal tract longer than LC705 during an intervention trial. The presence of mucus-binding pili on the surface of a nonpathogenic Gram-positive bacterial strain reveals a previously undescribed mechanism for the interaction of selected probiotic lactobacilli with host tissues.

Gut bacteria and health foods—the European perspective

Probiotics, prebiotics, and synbiotics aimed at improving intestinal health currently represent the largest segment of the functional foods market in Europe, Japan and Australia. Evidence continues to emerge demonstrating that these ingredients have the potential to improve human health in specific intestinal disorders. The European Commission, through its 5th Framework Programme, is presently focusing on a substantial effort in the science of the intestinal microbiota, its interaction with its host and methods to manipulate its composition and activity for the improvement of human health and well being. Eight multicentre and multidisciplinary research projects now cover a range of topics required for the development of efficacious probiotic foods, from understanding probiotic mechanisms at a molecular level; developing technologies to ensure delivery of stable products; and demonstrating safety and efficacy of specific probiotics in defined treatment targets. This concerted research effort promises to provide us with an enhanced understanding of the human intestinal microbiotas role in health and disease, and new approaches and products to tackle a variety of intestinal problems.

Bioprotectives and probiotics for dry sausages

The microbial stability of dry sausages is determined by the combination and timing of different factors referred to as the hurdle-concept. However, the hurdles present in dry sausage are not sufficient to prevent the survival of Listeria monocytogenes or enterohemorrhagic Escherichia coli O157:H7. Recently bioprotective lactic acid bacteria, which in addition to the production of antimicrobial lactic acid, have been found to contribute to the safety of the dry sausage by producing antimicrobial peptide, i.e. bacteriocins and other low-molecular-mass compounds. Furthermore, the possibilities to use probotics in dry sausage manufacturing process has been addressed recently. As one possible mode of action for probiotics is the production of antimicrobial compounds, lactic acid bacteria may act as both probiotic and bioprotective culture as well as fermenting agent in meat product, such as dry sausage.

Functional dairy products.

Introduction: classifying functional dairy products. Part 1 The health benefits of functional dairy products: Cancer Coronary heart disease Osteoporosis Probiotics and the management of food allergy Dairy products and the immune function in the elderly The therapeutic use of probiotics in gastrointestinal inflammation. Part 2 Functional dairy ingredients: Caseinophosphopeptides (CPPs) as functional ingredients Oligosaccharides Lactic acid bacteria (LAB) in functional dairy products Conjugated linoleic acid (CLA) as a functional ingredient. Part 3 Product development: Enhancing the functionality of prebiotics and probiotics Safety evaluation of probiotics Clinical trials Consumers and functional foods European research in probiotics and prebiotics: the PROEUHEALTH cluster The market for functional dairy products: the case of the United States.

Recovery of Lactobacillus rhamnosus GG from human colonic biopsies

The colonization of Lactobacillus rhamnosus GG (ATCC 53103, henceforth L.GG) in five human colonoscopy patients was studied. The test subjects consumed whey drink fermented with the bacterium for 12 d before the colonoscopy. The presence of L.GG was subsequently checked both in the faecal samples and in the colonic biopsies obtained from various locations in the large intestine. In all patients L.GG was the dominant faecal lactic acid bacterium as a result of the administration. In four patients L.GG could also be recovered from the biopsies, while with one patient (suffering from ulcerative colitis diagnosed during the colonoscopy) no L.GG was detected in the biopsy samples. The results suggest that L.GG is able to adhere in vivo to the colon. Study of the faecal samples alone is apparently not sufficient for elucidation of the gastrointestinal ecology of probiotic bacteria.

Development of functional ingredients for gut health

Abstract Microbial reactions in the gut have an essential role not only in gut health, but in general human health. The gut is the site of active fermentation of non-digestible diet components, as well as bioconversions and absorption of plant-derived compounds, such as phenolics. When developing nutritionally designed foods that promote health through gut microbial reactions, three different types of food ingredients can be used: living micro-organisms (probiotics), non-digestible carbohydrates (dietary fiber and prebiotics) and bioactive plant secondary metabolites (e.g. phenolics).