Elsevier

Bioresource Technology

Volume 102, Issue 3, February 2011, Pages 3309-3315
Bioresource Technology

Production of d-tagatose, a low caloric sweetener during milk fermentation using l-arabinose isomerase

https://doi.org/10.1016/j.biortech.2010.10.078Get rights and content

Abstract

Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus are used for the biotransformation of milk in yoghurt. During milk fermentation, these lactic acid bacteria (LAB) hydrolyze lactose producing a glucose moiety that is further metabolized and a galactose moiety that they are enable to metabolize. We investigated the ability of L. bulgaricus and S. thermophilus strains expressing a heterologous l-arabinose isomerase to convert residual d-galactose to d-tagatose. The Bacillus stearothermophilus US100 l-arabinose isomerase (US100 l-AI) was expressed in both LAB, using a new shuttle vector where the araA US100 gene is under the control of the strong and constitutive promoter of the L. bulgaricus ATCC 11842 hlbA gene. The production of l-AI by these LAB allowed the bioconversion of d-galactose to d-tagatose during fermentation in laboratory media and milk. We also established that the addition of l-AI to milk also allowed the conversion of d-galactose into d-tagatose during the fermentation process.

Introduction

Lactic acid bacteria (LAB) are of commercial importance as they are extensively used in the food industry as starters. Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) and Streptococcus thermophilus are among the widely used starters. They are used together to ferment milk into yoghurt (Tamine and Robinson, 1999) and some strains exhibit a cooperative growth (Hervé-Jimenez et al., 2009). Lactose is the unique sugar found in milk and it is the primary source of energy for LAB during milk fermentation. LAB have two systems for the transport and the metabolism of lactose: (i) a phosphoenolpyruvate (PEP) lactose phosphotransferase system (PTS) generally associated with a phospho-β-galactosidase system (Postma et al., 1993) and (ii) a lactose antiporter (LacS) generally coupled with a β-galactosidase (LacZ). The PEP-PTS system is conserved among numerous species including lactococci (Reizer et al., 1988) however, L. bulgaricus and S. thermophilus possess the second system based on the antiporter: lactose is brought into cell as a free sugar and is cleaved by a β-galactosidase into glucose and galactose (Leong-Morgenthaler et al., 1991). The glucose moiety is further metabolized to pyruvate which is used to produce lactate while the galactose moiety is exported outside the cell through the lactose antiporter in equimolar amounts with the transported lactose. Although these LAB species contain genes of the Leloir pathway which is the most ubiquitous route for galactose catabolism in these bacteria, this pathway is often non-functional in the dairy strains (De Vin et al., 2005, Hickey et al., 1986). In S. thermophilus, the galactose-minus phenotype is often due to a defect in the induction of the genes encoding the enzymes of the Leloir pathway during growth on lactose (De Vin et al., 2005, Turner and Martley, 1983). In L. bulgaricus ATCC 11842, the genome sequence revealed that the the genes involved in the Leloir pathway were indeed absent (Van de Guchte et al., 2006).

In yoghurt manufacturing, especially in the case of fruit or flavored products, sweetening compounds with high caloric value like sucrose are added to tone down the acidity of the product. Nowadays the demand for low caloric food products increases both to reduce caloric intake (for diet) and to reduce sugar consumption for instance in the case of diabetic patients, a disease which is rapidly increasing in developing countries as other metabolic disorders. In this work, we investigated whether the d-galactose produced during milk fermentation by LAB could be converted to d-tagatose, a caloric sweetener which could reduce the usual addition of high caloric sugars in some fermented product (Saelzer, 2005). d-Tagatose is a natural d-galactose isomer and is a rare ketohexose. The tagatose sweetening properties are similar to sucrose, but with a much lower caloric value as it is poorly degraded by the human body making it an interesting anti-hyperglycemiant agent (Levin et al., 1995, Livesey and Brown, 1996, Mazur, 1989, Zehner, 1994). As there is no abundant source of tagatose in nature, the unique economically robust process to produce this sweetener is the enzymatic isomerisation of d-galactose by the l-arabinose isomerase (l-AI) enzyme (Haltrich et al., 1998, Izumori et al., 1978, Jorgensen et al., 2004, Manzoni et al., 2001). This enzyme is found in several micro-organisms and isomerises in vivo the l-arabinose to l-ribulose (Kim et al., 2001). The l-AI of Bacillus stearothermophilus US100 (US100 l-AI) was previously characterized (Rhimi and Bejar, 2006): it is optimally active at pH 7.5 and 80 °C and in contrast with the previously characterized l-AIs it exhibits a low requirement for metal ions (Rhimi and Bejar, 2006).

In this work, we tested the ability of LAB strains producing US100 l-AI or engineered derivatives of this enzyme to convert d-galactose to d-tagatose during growth. The efficiency of d-tagatose bioconversion by the addition of the purified enzymes in milk was also investigated.

Section snippets

Bacterial strains and growth conditions

The bacterial strains and plasmids used in this work are listed in Table 1. Escherichia coli strains were grown in aerated Luria–Bertani medium (Difco) at 37 °C (Sambrook et al., 1989). Screening of E. coli recombinant clones having l-AI activity was achieved on McConkey agar medium, purchased from Sigma (Steinheim, Germany), supplemented with 1% l-arabinose (w/v) as a carbon source. L. bulgaricus and S. thermophilus strains were grown under anaerobic conditions at 42 °C in MRS medium (Difco) (De

Production of active US100 l-AI in yoghurt starters

To investigate the effect of the expression of the B. stearothermophilus US100 l-AI by yoghurt starters, the araA US100 gene encoding this enzyme was cloned in the pMR3 vector under the control of the strong and constitutive promoter of L. bulgaricus ATCC11842 hlbA gene (phlbA, Chouayekh et al., 2009), resulting in pMR4 (Table 1).

Plasmids pMR4 (phlbA:araA US100), pMR3 (phlbA without araA US100) and pMR2 (araA US100 without promoter) were transferred into the araA-minus HB101 E. coli strain. As

Conclusions

In this work the production of US100 l-AI by recombinant yoghurt starters was confirmed both in laboratory media and milk. In addition, we have demonstrated for the first time that in milk, the purified US100 l-AI and its derivatives efficiently converted d-galactose into d-tagatose leading to sweetener concentrations reported as sufficient to modify the yoghurt taste. Our data also evidenced that the biochemical properties of the enzyme impact the efficiency of the process; we will further

Acknowledgements

This research was funded by the Tunisian Government “Contrat Programme CBS-LEMP” and the International Foundation for Science, Stockholm, Sweden, through a grant to Dr. Hichem CHOUAYEKH (research grant agreements N°E/4126-1&2). Part of the work was also supported by the CMCU project (2007–2009) n°07G0922 “CHOUAYEKH/MAGUIN”. Moez RHIMI received a financial support from INRA (MICA division). The authors acknowledge Samira Boudebbouze, Dr. Michel JUY and Dr. Luciana Hervé-Jimenez for their help as

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