Structure determination of the neutral exopolysaccharide produced by Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1
Graphical abstract
Introduction
Dairy starters producing exopolysaccharides (EPSs) have been used as syneresis inhibitors or for texture improvement of fermented products for a long time. Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus are generally used for the production of yogurt. EPS-producing strains exist within both species.1, 2 Lactobacillus bulgaricus is a thermophilic Gram-positive lactic acid bacterium used in the production of fermented milk such as yogurt. EPSs produced by L. bulgaricus presently used in the dairy industry are mainly neutral.
Strain OLL1073R-1 has been shown to produce EPSs with immunomodulatory effects.3, 4, 5, 6, 7, 8 The oral administration of yogurt fermented with this bacterial strain or its EPSs to mice has been shown to enhance natural killer (NK) cell activity and exert anti-influenza virus activity.6, 8 The ingestion of yogurt fermented with strain OLL1073R-1 has been reported to reduce the risk of catching the common cold and flu and also to augment NK cell activity in subjects with low NK cell activity.7 Total EPS could be fractionated into neutral polysaccharide (NPS) and anionic polysaccharide (APS). The latter contains phosphate, which is required for activity.3 Both NPS and APS have been partially characterized,9 but their structure has never been determined.
In this work, we report physicochemical properties of the NPS produced by L. delbrueckii subsp. bulgaricus strain OLL1073R-1 and unambiguously determined its primary sequence. The repeating unit structure will be compared to that of other strains of the same species.
Section snippets
Yields and quality controls
L. delbrueckii subsp. bulgaricus strain OLL1073-1 was cultivated for 18 h at 37 °C in 10% reconstituted skim milk, and ethanol precipitation afforded 1546 mg of crude EPS containing 86% NPS, 13% APS, and 1% protein. After DEAE Sepharose purification, 1260 mg of NPS (0.13% protein) and 34.1 mg of APS (0.9% protein) were obtained from 10 kg of total cell culture.
Elemental analysis of the NPS revealed the presence of carbon and hydrogen only.
Physicochemical properties
Physicochemical characterization of the NPS was
Discussion
The structure of the NPS from strain OLL1073R-1 is different from that of the six unique L. bulgaricus EPS structures reported in the literature (Fig. 5).16, 17, 18, 19, 20, 21, 22, 23, 24 There are however some common elements: the repeating unit contains five sugars, as for three other strains (291, LBB.B26, and LBB.B332); only glucose and galactose are present, again as in three EPSs (291, NCFB 2074, and LBB.B26); the EPSs of two other strains (EU23 and LBB.B26) similarly possess a short
Production and purification
The preparation of OLL1073R-1 EPS has been reported by Uemura et al.9 Briefly, L. delbrueckii subsp. bulgaricus OLL1073R-1 was incubated in 10% skimmed milk at 37 °C for 18 h. The whole culture (10 kg) was treated with trichloroacetic acid (TCA) (10% final) and precipitated denatured proteins and bacterial cells were removed by centrifugation at 12,000×g for 20 min. The supernatant was treated with an equal volume of cold ethanol, kept at 4 °C overnight, and centrifugation was performed as
Acknowledgements
We thank Dr. Hassan Sabik for ESI–MS spectra.
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Present address: Department of Biotechnology and Environmental Engineering, Faculty of Engineering, Hachinohe Institute of Technology, 88-1 Obiraki, Myo Hachinohe, Aomori 031-8501, Japan.