Urease production by Streptococcus thermophilus

doi:10.1016/j.fm.2007.07.001

Food Microbiology

Volume 25, Issue 1, February 2008, Pages 113-119

https://doi.org/10.1016/j.fm.2007.07.001 Get rights and content

Abstract

In order to identify potential alternative sources of urease for the removal of urea from alcoholic beverages, 205 strains of lactic acid bacteria belonging to 27 different species were screened for urease production. Only Streptococcus thermophilus produced urease. Cell permeabilization with toluene allowed to increase activity significantly. Optimal pH for urease activity in whole and permeabilized cells and of cell free extracts differed slightly, but was in the range 6.0–7.0. Significant activity was retained at pH 3.0 and 8.0, and, for cell free extracts, at pH 4.0 in the presence of ethanol. Urease production was evaluated in fermentations with pH control (5.25–6.5) and without pH control. Very little urease was produced in absence of urea, which at 5 g/l slowed growth significantly in fermentations without pH control, but prevented a decrease in pH below 5.1 and resulted in higher final biomass. Optimal pH for growth was between 6.0 and 6.5 but specific urease activity was higher for fermentations at low pH at the beginning of the exponential phase. However, a higher total urease activity was obtained at pH 6.0 and 6.5 because of higher biomass. Potential technological applications of urease production by S. thermophilus are discussed.

Introduction

Urease (urea amidohydrolase, EC 3.5.1.15) catalyzes the hydrolysis of urea to ammonia and carbamate, which spontaneously hydrolyzes to give a further molecule of ammonia and carbonate. Urease production is widespread in prokaryotes: all bacterial ureases are nickel metalloproteins and share significant sequence homology among them and with eucaryotic ureases (Mobley et al., 1995). Regulation of the expression of the urease operon varies in different species. In oral lactic acid bacteria (LAB), like Streptococcus salivarius, it is affected by the presence of urea, pH and sugar availability (Chen and Burne, 1996; Chen et al., 1998). This is in good agreement with the dual role of ureases in response to acid stress and nitrogen metabolism (Chen et al., 2000). In the human pathogen Helicobacter pylori urease is the most important factor contributing to the maintenance of intracellular and periplasmic pH near neutrality under acid stress (Pflock et al., 2006) and is essential for colonization of several animal models.

Acid ureases with pH optima between 2.0 and 4.5 (Mobley et al., 1995) are produced by some species of lactic acid bacteria (Kakimoto et al., 1990a, Kakimoto et al., 1989, Kakimoto et al., 1990b; Yamazaki et al., 1990) and corynebacteria (Miyagawa et al., 1999) and are potentially useful for the reduction of urea content in wine and in other alcoholic beverages, like sake, thus reducing the risk of accumulation of ethyl carbamate (ETC; Kodama, 1996; Butzke and Bisson, 1997; Fidaleo et al., 2006). ETC is a potential carcinogenic agent which is spontaneously produced in wine and other alcoholic beverages in reactions between urea or other compounds containing carbamylic groups (citrulline and carbamyl phosphate) and ethanol (Ough et al., 1988). An acid urease produced by Lactobacillus fermentum is commercialized by several producers (Fidaleo et al., 2006) and the European Commission has expressed a positive opinion on its use in wine (http://europa.eu.int/comm/food/fs/sc/scf/out19_en.html).

Among urease-producing species, Streptococcus thermophilus is particularly interesting. It is a moderately thermophilic, food-grade, industrially important microorganism which is used as a starter in the production of fermented milks and cheese and is commonly isolated from artisanal cheeses and natural starter cultures (Parente and Cogan, 2004). Urease production is common in S. thermophilus (Mora et al., 2002) and, although it is important for acid stress resistance (Mora et al., 2005), it slows pH decrease in milk and cheese due to the production of ammonia (Mora et al., 2004; Monnet et al., 2004). The urease operon of S. thermophilus has been recently characterized (Mora et al., 2004) and it has been found to be similar to that of the taxonomically related S. salivarius (Chen and Burne, 1996; Chen et al., 1998).

In order to find alternatives to the commercially available acid ureases, we screened lactic acid bacteria obtained from several food sources. A preliminary study was then conducted on urease-producing S. thermophilus to obtain information on conditions for urease production and urease activity as a function of pH.

Section snippets

Microbial strains

Strains of lactic acid bacteria (205) isolated from milk and dairy products (130), from sourdoughs (41) or obtained from culture collections (34), belonging to different species of the genera Enterococcus (3 E. faecalis strains, 2 E. faecium, 2 E. pseudoavium), Lactobacillus (1 L. alimentarius, 2 L. brevis, 7 L. casei, 2 L. coryniformis, 10 L. curvatus, 1 L. farciminis, 3 L. fermentum, 11 L. helveticus, 22 L. paracasei, 6 L. paraplantarum, 2 L. pentosus, 25 L. plantarum, 6 L. rhamnosus, 2 L.

Results

Out of 205 strains belonging to 27 species of LAB screened for urease production only 34 S. thermophilus strains (79% of the strains belonging to this species) scored positive. Urease activity was measured for whole cells, culture broth supernatants and cell lysates of the urease-positive strains in a quantitative assay. The activity was always intracellular (no measurable activity was found in cell-free supernatants; data not shown). A large variability was observed and urease activity of

Discussion

Although urease production is widespread among prokaryotes (Mobley et al., 1995), with the exception of S. thermophilus (Mora et al., 2002, Mora et al., 2004) and S. salivarius (Chen and Burne, 1996; Chen et al., 1996, Chen et al., 2000, Chen et al., 1998) only a few species of lactic acid bacteria have been tested for urease activity and found positive (Kakimoto et al., 1990a, Kakimoto et al., 1989, Kakimoto et al., 1990b; Yamazaki et al., 1990). Acid urease activity has been found in L.

Conclusions

Urease production by S. thermophilus has been regarded as detrimental for its activity as starter culture in yoghurt and in cheese production (Mora et al., 2004; Monnet et al., 2004) because milk acidification is delayed by the presence of urea. However, growth in the presence of urease, although slower, results in higher cell numbers and may reduce cell damage at acid pH, thus resulting in higher activity. Moreover, urea can be used to keep pH constant during lactic acid fermentation. A

Acknowledgments

This work was partly supported by a Post-Doc grant to Teresa Zotta from Università degli Studi della Basilicata and by a grant from Ministero dell’Università e della Ricerca Scientifica, Rome (PRIN2005 Grant 2005071833). The skilled technical assistance of Antonella Tauriello and Giancarlo Vaccaro is gratefully acknowledged.

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Urease production by Streptococcus thermophilus

Abstract

Introduction

Section snippets

Microbial strains

Results

Discussion

Conclusions

Acknowledgments

Int. J. Food Microbiol.

FEMS Microbiol. Lett.

Int. Dairy J.

Proc. Biochem.

J. Biotechnol.

J. Dairy Sci.

Res. Microbiol.

Enzyme Microbial. Technol.

J. Biotechnol.

J. Microbiol. Methods

Streptococcus salivarius urease: genetic and biochemical characterization and expression in a dental plaque Streptococcus

Infect. Immun.

Transcriptional regulation of the Streptococcus salivarius 57.I urease operon

J. Bacteriol.

Dual functions of Streptococcus salivarius urease

J. Bacteriol.