Maria Saarela

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.

Analysis of the Fecal Microbiota of Irritable Bowel Syndrome Patients and Healthy Controls with Real-Time PCR

OBJECTIVE:The gut microbiota may contribute to the onset and maintenance of irritable bowel syndrome (IBS). In this study, the microbiotas of patients suffering from IBS were compared with a control group devoid of gastrointestinal (GI) symptoms.METHODS:Fecal microbiota of patients (n = 27) fulfilling the Rome II criteria for IBS was compared with age- and gender-matched control subjects (n = 22). Fecal samples were obtained at 3 months intervals. Total bacterial DNA was analyzed by 20 quantitative real-time PCR assays covering approximately 300 bacterial species.RESULTS:Extensive individual variation was observed in the GI microbiota among both the IBS- and control groups. Sorting of the IBS patients according to the symptom subtypes (diarrhea, constipation, and alternating predominant type) revealed that lower amounts of Lactobacillus spp. were present in the samples of diarrhea predominant IBS patients wheras constipation predominant IBS patients carried increased amounts of Veillonella spp. Average results from three fecal samples suggested differences in the Clostridium coccoides subgroup and Bifidobacterium catenulatum group between IBS patients (n = 21) and controls (n = 15). Of the intestinal pathogens earlier associated with IBS, no indications of Helicobacter spp. or Clostridium difficile were found whereas one case of Campylobacter jejuni was identified by sequencing.CONCLUSIONS:With these real-time PCR assays, quantitative alterations in the GI microbiota of IBS patients were found. Increasing microbial DNA sequence information will further allow designing of new real-time PCR assays for a more extensive analysis of intestinal microbes in IBS.

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.

Bifidobacterial Diversity in Human Feces Detected by Genus-Specific PCR and Denaturing Gradient Gel Electrophoresis

ABSTRACT We describe the development and validation of a method for the qualitative analysis of complex bifidobacterial communities based on PCR and denaturing gradient gel electrophoresis (DGGE).Bifidobacterium genus-specific primers were used to amplify an approximately 520-bp fragment from the 16S ribosomal DNA (rDNA), and the fragments were separated in a sequence-specific manner in DGGE. PCR products of the same length from different bifidobacterial species showed good separation upon DGGE. DGGE of fecal 16S rDNA amplicons from five adult individuals showed host-specific populations of bifidobacteria that were stable over a period of 4 weeks. Sequencing of fecal amplicons resulted in Bifidobacterium-like sequences, confirming that the profiles indeed represent the bifidobacterial population of feces. Bifidobacterium adolescentis was found to be the most common species in feces of the human adult subjects in this study. The methodological approach revealed intragenomic 16S rDNA heterogeneity in the type strain of B. adolescentis, E-981074. The strain was found to harbor five copies of 16S rDNA, two of which were sequenced. The two 16S rDNA sequences of B. adolescentis E-981074T exhibited microheterogeneity differing in eight positions over almost the total length of the gene.

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.

Contribution of diet to the composition of the human gut microbiota.

In the human gut, millions of bacteria contribute to the microbiota, whose composition is specific for every individual. Although we are just at the very beginning of understanding the microbiota concept, we already know that the composition of the microbiota has a profound impact on human health. A key factor in determining gut microbiota composition is diet. Preliminary evidence suggests that dietary patterns are associated with distinct combinations of bacteria in the intestine, also called enterotypes. Western diets result in significantly different microbiota compositions than traditional diets. It is currently unknown which food constituents specifically promote growth and functionality of beneficial bacteria in the intestine. The aim of this review is to summarize the recently published evidence from human in vivo studies on the gut microbiota-modulating effects of diet. It includes sections on dietary patterns (e.g. Western diet), whole foods, food constituents, as wells as food-associated microbes and their influence on the composition of human gut microbiota. The conclusions highlight the problems faced by scientists in this fast-developing field of research, and the need for high-quality, large-scale human dietary intervention studies.

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.

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).

Recommendations for the viability assessment of probiotics as concentrated cultures and in food matrices.

Due to the fact that probiotic cells need to be alive when they are consumed, culture-based analysis (plate count) is critical in ascertaining the quality (numbers of viable cells) of probiotic products. Since probiotic cells are typically stressed, due to various factors related to their production, processing and formulation, the standard methodology for total plate counts tends to underestimate the cell numbers of these products. Furthermore, products such as microencapsulated cultures require modifications in the release and sampling procedure in order to correctly estimate viable counts. This review examines the enumeration of probiotic bacteria in the following commercial products: powders, microencapsulated cultures, frozen concentrates, capsules, foods and beverages. The parameters which are specifically examined include: sample preparation (rehydration, thawing), dilutions (homogenization, media) and plating (media, incubation) procedures. Recommendations are provided for each of these analytical steps to improve the accuracy of the analysis. Although the recommendations specifically target the analysis of probiotics, many will apply to the analysis of commercial lactic starter cultures used in food fermentations as well.