Z Lebensm Unters Forsch A (1999) 208 : 57–59
Q Springer-Verlag 1999
ORIGINAL PAPER
Marisa S. Garro 7 Graciela F. de Valdez Guillermo Oliver 7 Graciela S. de Giori
Hydrolysis of soya milk oligosaccharides by Bifidobacterium longum CRL 849
Received: 29 December 1997
Abstract The reduction of soya milk oligosaccharides by Bifidobacterium longum CRL 849 was studied. The utilization of stachyose was concomitant with the use of sucrose. Maximum hydrolysis of stachyose (49.3%) occurred during the first 7 h of incubation at 37 7C, while a 79.3% decrease in the concentration of sucrose was observed after 9 h. No raffinose was detected after hydrolysis of the stachyose. Cell population decreased after 8 h of incubation because of the low pH attained (pH 4.7). L(c)-Lactate concentration was higher than acetate (molar ratio 6.7 : 1) at 6 h followed by a slow increase in acetate formation. Ethanol was detected in small amounts at the end of the incubation time (24 h). Key words Bifidobacterium 7 Soya milk 7 Oligosaccharides
Introduction Soya milk (SM) is considered a suitable and economical substitute for cow’s milk and an ideal nutritional supplement for lactose-intolerant individuals [1]. However, the consumption of SM may lead to digestive problems associated with the presence of raffinose and stachyose. These sugars are degraded by the human intestinal bacteria to carbon dioxide, hydrogen and methane, producing flatulence and abdominal pains [2]. Therefore, processes including fermentation of SM with fungi [3, 4] or lactic acid bacteria [5] have been
M.S. Garro 7 C.F. de Valdez 7 C. Oliver 7 G.S. de Giori (Y) Centro de Referencia para Lactobacilos (CERELA), Chacabuco 145, 4000 Tucumán, Argentina e-mail:
[email protected] G.F. de Valdez 7 G.S. de Giori Cátedra de Microbiología Superior, Facultad de Bioquímica, Química y Farmacia, Universidad Nacional de Tucumán, Argentina
examined as means to avoid such problems and to improve the acceptability of this substrate. In this respect bifidobacteria are thought to play an important role in the host’s health [6]. These micro-organisms are very useful from a nutritional standpoint, and it has been reported that certain indigestible saccharides are useful for promoting the growth of bifidobacteria [7]. The present work was undertaken to evaluate the ability of Bifidobacterium longum CRL 849 to reduce SM galactosaccharides for probiotic purposes.
Materials and methods Micro-organism and growth conditions. B. longum CRL 849 was obtained from the culture collection of the Centro de Referencia para Lactobacilos (CERELA), Argentina. The SM used as growth medium had the following approximate composition: 3.0% protein, 2.5% lipid, 3.5% sugar, 0.5% ash, 90.5% water. The sugar fraction comprised 2.8% sucrose and 0.7% stachyose. The SM was sterilized prior to use at 115 7C for 20 min and had a final pH of 6.8–7.0. Fermentation. Growth experiments were performed in Erlenmeyer flasks containing 500 ml SM. Freeze-dried preparations [8] of B. longum CRL 849 were used as an inoculum at a level of 1.5!10 7 colony forming units (cfu) ml P1 (final concentration). The cultures were incubated statically at 37 7C for 24 h. Samples were taken at 0 h, 3 h, 6 h, 9 h and 24 h and tested for pH and cell viability. For evaluation of residual sugar’s, lactic acid, acetic acid and ethanol, samples were frozen at P20 7C until analysis. Uninoculated SM treated in the same way was used as a control. Cell viability was determined by the plate dilution method using TPY agar [9]. Serial dilutions of each sample were plated in duplicate, and the plates were incubated anaerobically at 37 7C for 72 h. The resulting colonies were measured in colony forming units per millilitre. Analytical assays. The levels of stachyose, sucrose and their hydrolysis products (raffinose, galactose, glucose and fructose) were determined in SM cultures by high-performance liquid chromatography (HPLC) on a Gilson system (Villers le Bel, France) coupled to a differential refractometer (LKB, model 2142, Bromma, Sweden) and a registrator (Shimadzu, model CR 601; Chromatopac). Samples were deproteinized [5] before sugar determination. The supernatants obtained were injected into the column through a 20 ml sample loop (Reodyne, model 9125). HPLC was perform-
58 ed on a REZEX RHM monosaccharides column (300!7.8 mm) (Phenomenex, Torrance, Calif.) at a column temperature of 55 7C. The mobile phase was HPLC-grade bi-distilled water and the flow rate was 0.6 ml min P1. External standards were prepared by diluting specific amounts of sugars in deionized water. Standard curves were constructed for each carbohydrate. Least-squares regression analysis was used to derive equations from the values reported for each carbohydrate. These equations were used to calculate carbohydrate consumption in the SM. L(c)-Lactate was determined enzymatically according to Gutmann and Wahlefeld [10] and D(P)-lactate according to Gawehn and Bergmeyer [11]. Determination of acetic acid and ethanol was performed on a gas chromatograph (Gow Mac Instrument Co.) equipped with a flame ionization detector. Separations took place in a stainless steel capillary column (30 m!0.32 mm i.d.) packed with ATWAX (0.25 mm) (Heliflex capillary, Alltech). Helium was used as the carrier gas. The temperature of the column was programmed at 90 7C (isothermal), the injector temperature at 130 7C and the detector temperature at 190 7C. Data were analysed using an integrator (Data Jet, Spectra-Physics) and quantification was achieved by internal standard calibration. The pH of the samples was measured by potentiometric methods. Reproducibility. All results presented in this paper are the means of three assays. The maximum variation from the mean values was less than 5%.
Results and discussion B. longum CRL 849 showed a maximal cell density (1.5!10 9 cfu ml P1) in SM after 8 h of incubation with a specific growth rate (m) of 0.62 h P1, followed by a slight decrease (1.5 log cycles) at 24 h of fermentation. The drop in pH was more pronounced within the first 6 h of incubation, coincidentally with the highest rate of carbohydrate breakdown (Fig. 1a). The pH attained at the end of fermentation was 4.3. Rasic and Kurmann [12] consider pH values of 4.2–4.5 to be a minimum for the survival of B. longum. The acid developed by the cells was probably the factor that limited growth and sugar fermentation in SM (see below). The kinetics of both substrate disappearance and end-product formation during fermentation of SM with B. longum CRL 849 are shown in Fig. 1b–c. Sucrose was preferentially hydrolysed (21.7% of residual carbohydrate was found after 9 h of fermentation) whereas 50.7% of the original levels of stachyose were detected (Fig. 1b). After this time, the metabolism of this sugar stopped, probably due to an inactivation of a-galactosidase (the enzyme responsible for the hydrolysis of stachyose) as a consequence of the acidity developed by the cultures [13]. It is interesting to note that raffinose, as an intermediate product of stachyose catabolism, was not detected, differing-sharply from the results reported by Garro et al. [14] for Lactobacillus fermentum CRL 251. Apparently, a-galactosidase from these organisms will have a different specificity on the substrate stachyose (Fig. 2). In B. longum CRL 849, the enzyme will act on both a-(1,6)-galactoside bonds of stachyose rel-
Fig. 1a–c Fermentation patterns of Bifidobacterium longum in soya milk: a (x) viable counts; (L) pH; b Residual carbohydrates: ([) stachyose, (}) raffinose, (n) sucrose, (m) galactose, (l) glucose, (N) fructose; c End-products: (n) L(c)-lactate, (}) acetate, ([) ethanol
easing galactose and sucrose, and an invertase will hydrolyse the b-(1,2)-fructofuranoside bond of this disaccharide releasing fructose and glucose. When both enzymes act simultaneously, the total degradation of sta-
Fig. 2 Structure of stachyose. a a-(1,6)-galactoside bound (enzyme: a-galactosidase); b b-(1,2)-fructofuranoside bound (enzyme: invertase)
59
chyose occurs. However, the monosaccharides glucose, galactose and fructose were scarcely detected, which suggests that they were consumed by the micro-organism during growth (Fig. 1b). The end-product formation as a result of microbial metabolism of B. longum CRL 849 in SM is shown in Fig. 1c. The greatest production of L(c)-lactate (2.83 g l P1) (D(P)-lactate was not found) took place between 3 h and 6 h of incubation. During this time, very small amounts of acetic acid (0.28 g l P1) were detected. The synthesis of ethanol began after 6 h and increased steadily at the same rate as acetate production. The molar ratio lactate : acetate changed throughout the fermentation period, being 2.4 : 1.0 after 24 h. These results are similar to those reported by Desjardins et al. [15] for a strain of B. bifidum in milk which produced 2.5 times more lactate than acetate. It is well known that Bifidobacterium growing on glucose produces lactate and acetate in a molar ratio 2 : 3 [12]. However, modifications in the culture conditions may result in a change in this ratio. Thus, the SM substrate and aerobic conditions might explain the end-product profile found in B. longum CRL 849. The levels of stachyose reduction found in this study show that Bifidobacterium metabolized the tetrasaccharide faster than Lactobacillus plantarum B-246 [5], which required 30 h of fermentation to reach similar values of residual stachyose. Most of the data currently available on the growth characteristics and organic acid formation kinetics of bifidobacteria have been carried out in MRS and TPY media [9, 16]. In this study, it is shown that the endproduct profile of B. longum CRL 849 grown in SM was affected by the incubation time and the acidity conditions. From a practical point of view, a 6 h incubation with this strain is enough to assure 50% stachyose reduction (the main flatulence producer oligosaccharide) and to achieve a cell population of 1.0!10 9 cfu ml P1 in the final product. Thus, fermentation of SM substrate
by B. longum CRL 849 should be considered as a suitable alternative for improving galactosaccharide digestion when bioprocessed soya bean products are included in the human diet. Acknowledgements This work was supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Consejo de Ciencia y Técnica de la Universidad Nacional de Tucumán (CIUNT), and Agencia Nacional de Promoción Científica y Tecnológica (FONCYT), Argentina.
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