Jeroen Hugenholtz

An overview of the functionality of exopolysaccharides produced by lactic acid bacteria

Abstract Lactic acid bacteria (LAB) that produce exopolysaccharides (EPSs) play an important role in the dairy industry because of their contribution to the consistency and rheology of fermented milk products. The EPS polymers can be considered as natural biothickeners because they are produced in situ by the LAB-starters that have General Recognised As Safe status (GRAS). The physico-chemical properties of EPSs determine their viscosifying efficiency. Hence, the knowledge of the structure–function relationship of these biopolymers is crucial in order to choose or design polymers for a specific technological application. In addition, health benefits have been attributed to some of these EPSs, particularly antitumor and immunomodulating activities. Also a prebiotic role has been suggested. However, almost all studies were performed in vitro and scarce information is available concerning in vivo experiments with oral administration. This overview focuses on the recent information about the functional properties of lactic acid bacterial EPSs, including both technological and health-promoting aspects.

Microbes from raw milk for fermented dairy products

Milk has a high nutritive value, not only for the new-born mammal and for the human consumer, but also for microbes. Raw milk kept at room temperature will be liable to microbial spoilage. After some days, the milk will spontaneously become sour. This is generally due to the activity of lactic acid bacteria. A flora of these bacteria may develop, which can be transferred deliberately to fresh milk in order to maintain or even strengthen it. This principle is the basis for controlled acidification of milk towards products, sustainable and safe, with most often an attractive flavour. Various types of fermented milks and derived products have been developed in all parts of the world, each with its own characteristic history. Their nature depends very much on the type of milk used, on the pre-treatment of the milk, on the temperature (climate) and the conditions of fermentation and on the subsequent technological treatments. Most fermented dairy products contain lactic acid bacteria, but other bacteria, yeasts and moulds may be involved as well. In optimising the manufacturing processes, starter cultures for fermented dairy products have been developed. They are composed of selected microorganisms, propagated as multiple-strain starters consisting of a defined mixture of pure cultures or as mixed-strain starters consisting of an undefined mixture of different types of bacteria. The use of starters, on the one hand, has been tremendously positive with respect to the quality of the product, but, on the other hand, it has diminished the diversity of fermented dairy products. Since the dairy industry is keen to explore new possibilities for enhancing the diversity of its product range, there is a new interest nowadays in searching for potential starter organisms from the pool, which existed at the time of raw milk fermentation. This contribution reviews some potential opportunities and recent developments in this search.

Conversion of Lactococcus lactis from homolactic to homoalanine fermentation through metabolic engineering

We report the engineering of Lactococcus lactis to produce the amino acid l-alanine. The primary end product of sugar metabolism in wild-type L. lactis is lactate (homolactic fermentation). The terminal enzymatic reaction (pyruvate + NADH→l-lactate + NAD+) is performed by l-lactate dehydrogenase (l-LDH). We rerouted the carbon flux toward alanine by expressing the Bacillus sphaericus alanine dehydrogenase (l-AlaDH; pyruvate + NADH + NH4+→l-alanine + NAD+ + H2O). Expression of l-AlaDH in an l-LDH-deficient strain permitted production of alanine as the sole end product (homoalanine fermentation). Finally, stereospecific production (>99%) of l-alanine was achieved by disrupting the gene encoding alanine racemase, opening the door to the industrial production of this stereoisomer in food products or bioreactors.

Effects of Cultivation Conditions on Folate Production by Lactic Acid Bacteria

ABSTRACT A variety of lactic acid bacteria were screened for their ability to produce folate intracellularly and/or extracellularly. Lactococcus lactis, Streptococcus thermophilus, and Leuconostoc spp. all produced folate, while most Lactobacillus spp., with the exception of Lactobacillus plantarum, were not able to produce folate. Folate production was further investigated in L. lactis as a model organism for metabolic engineering and in S. thermophilus for direct translation to (dairy) applications. For both these two lactic acid bacteria, an inverse relationship was observed between growth rate and folate production. When cultures were grown at inhibitory concentrations of antibiotics or salt or when the bacteria were subjected to low growth rates in chemostat cultures, folate levels in the cultures were increased relative to cell mass and (lactic) acid production. S. thermophilus excreted more folate than L. lactis, presumably as a result of differences in the number of glutamyl residues of the folate produced. In S. thermophilus 5,10-methenyl and 5-formyl tetrahydrofolate were detected as the major folate derivatives, both containing three glutamyl residues, while in L. lactis 5,10-methenyl and 10-formyl tetrahydrofolate were found, both with either four, five, or six glutamyl residues. Excretion of folate was stimulated at lower pH in S. thermophilus, but pH had no effect on folate excretion by L. lactis. Finally, several environmental parameters that influence folate production in these lactic acid bacteria were observed; high external pH increased folate production and the addition of p-aminobenzoic acid stimulated folate production, while high tyrosine concentrations led to decreased folate biosynthesis.

Increased Production of Folate by Metabolic Engineering of Lactococcus lactis

ABSTRACT The dairy starter bacterium Lactococcus lactis is able to synthesize folate and accumulates large amounts of folate, predominantly in the polyglutamyl form. Only small amounts of the produced folate are released in the extracellular medium. Five genes involved in folate biosynthesis were identified in a folate gene cluster in L. lactis MG1363: folA, folB, folKE, folP, and folC. The gene folKE encodes the biprotein 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase and GTP cyclohydrolase I. The overexpression of folKE in L. lactis was found to increase the extracellular folate production almost 10-fold, while the total folate production increased almost 3-fold. The controlled combined overexpression of folKE and folC, encoding polyglutamyl folate synthetase, increased the retention of folate in the cell. The cloning and overexpression of folA, encoding dihydrofolate reductase, decreased the folate production twofold, suggesting a feedback inhibition of reduced folates on folate biosynthesis.

Unraveling microbial interactions in food fermentations; from classical to genomics approaches

Fermentation, the microbial degradation of organic compounds without net oxidation, is an important process in the global carbon cycle and is also exploited worldwide for the production and preservation of food. It is one of the oldest food-processing technologies known, with some records dating back to 6,000 b.c. (50). The link between food and microbiology was laid by Pasteur, who found that yeasts were responsible for alcoholic fermentation (106). Since that discovery, scientific and industrial interests in food microbiology started to grow and continue to increase today. The number of food products that rely on fermentation in one or more steps of their production is tremendous. They form an important constituent of the daily diet and rank among the most innovative product categories in the food industry. Most of the important microorganisms applied in the production of fermented foods have been studied for decades, yielding a wealth of information on their physiology and genetics in relation to product functionalities, such as the development of flavor, taste, and texture. The recent emergence of genomics has opened new avenues for the systematic analysis of microbial metabolism and the responses of microorganisms to their environment. Additionally, genomics has boosted research on important food microbes (22, 90, 93). Much of this research focuses on the performance of a single strain, including its interactions with the food matrix. However, food fermentations are typically carried out by mixed cultures consisting of multiple strains or species. Population dynamics play a crucial role in the performance of mixed-culture fermentations, and for many years, studies on mixed-culture food fermentations have focused on analyzing population dynamics using classical and molecular methods. Many of these studies are mainly descriptive, and relatively little is known about the mechanisms governing population dynamics in general and the molecular interactions that occur between the consortium members in particular. The availability of genome sequences for several species that are of industrial importance as well as technological advances in functional genomics enable new approaches to study food microbiology beyond the single species level and allow an integral analysis of the interactions and metabolic activity in mixed cultures. Here we review the current knowledge on important food fermentation processes, focusing on the bacterial interactions. In addition, we illustrate how genomics approaches may contribute to the elucidation of the interaction networks between microbes, including interactions with the food environment. This information may find application in the industry through rational optimization and increased control over mixed-culture fermentations.

Lactobacillus reuteri CRL1098 Produces Cobalamin

We found that Lactobacillus reuteri CRL1098, a lactic acid bacterium isolated from sourdough, is able to produce cobalamin. The sugar-glycerol cofermentation in vitamin B(12)-free medium showed that this strain was able to reduce glycerol through a well-known cobalamin-dependent reaction with the formation of 1,3-propanediol as a final product. The cell extract of L. reuteri corrected the coenzyme B12 requirement of Lactobacillus delbrueckii subsp. lactis ATCC 7830 and allowed the growth of Salmonella enterica serovar Typhimurium (metE cbiB) and Escherichia coli (metE) in minimal medium. Preliminary genetic studies of cobalamin biosynthesis genes from L. reuteri allowed the identification of cob genes which encode the CobA, CbiJ, and CbiK enzymes involved in the cobalamin pathway. The cobamide produced by L. reuteri, isolated in its cyanide form by using reverse-phase high-pressure liquid chromatography, showed a UV-visible spectrum identical to that of standard cyanocobalamin (vitamin B12).

Riboflavin Production in Lactococcus lactis: Potential for In Situ Production of Vitamin-Enriched Foods

ABSTRACT This study describes the genetic analysis of the riboflavin (vitamin B2) biosynthetic (rib) operon in the lactic acid bacterium Lactococcus lactis subsp. cremoris strain NZ9000. Functional analysis of the genes of the L. lactis rib operon was performed by using complementation studies, as well as by deletion analysis. In addition, gene-specific genetic engineering was used to examine which genes of the rib operon need to be overexpressed in order to effect riboflavin overproduction. Transcriptional regulation of the L. lactis riboflavin biosynthetic process was investigated by using Northern hybridization and primer extension, as well as the analysis of roseoflavin-induced riboflavin-overproducing L. lactis isolates. The latter analysis revealed the presence of both nucleotide replacements and deletions in the regulatory region of the rib operon. The results presented here are an important step toward the development of fermented foods containing increased levels of riboflavin, produced in situ, thus negating the need for vitamin fortification.

Metabolic engineering of lactic acid bacteria for the production of nutraceuticals

Lactic acid bacteria display a relatively simple and well-described metabolism where the sugar source is converted mainly to lactic acid. Here we will shortly describe metabolic engineering strategies on the level of sugar metabolism, that lead to either the efficient re-routing of the lactococcal sugar metabolism to nutritional end-products other than lactic acid such as L-alanine, several low-calorie sugars and oligosaccharides or to enhancement of sugar metabolism for complete removal of (undesirable) sugars from food materials. Moreover, we will review current metabolic engineering approaches that aim at increasing the flux through complex biosynthetic pathways, leading to the production of the B-vitamins folate and riboflavin. An overview of these metabolic engineering activities can be found on the website of the Nutra Cells 5th Framework EU-project (www.nutracells.com). Finally, the impact of the developments in the area of genomics and corresponding high-throughput technologies on nutraceutical production will be discussed.

Physiological function of exopolysaccharides produced by Lactococcus lactis.

The physiological function of EPS produced by Lactococcus lactis was studied by comparing the tolerance of the non-EPS producing strain L. lactis ssp. cremoris MG1614 and an EPS producing isogenic variant of this strain to several anti-microbial factors. There was no difference in the sensitivity of the strains to increased temperatures, freezing or freeze-drying and the antibiotics, penicillin and vancomycin. A model system showed that EPS production did not affect the survival of L. lactis during passage through the gastrointestinal tract although the EPS itself was not degraded during this passage. The presence of cell associated EPS and EPS in suspension resulted in an increased tolerance to copper and nisin. Furthermore, cell associated EPS also protected the bacteria against bacteriophages and the cell wall degrading enzyme lysozyme. However, it has not been possible, so far, to increase EPS production using the presence of copper, nisin, lysozyme or bacteriophages as inducing factors.