Introduction

The term “oligosaccharides” is broadly used for saccharides having the degree of polymerization of 2–10 (Nakakuki 1993). Structurally, Galacto oligosaccharides are composed of 2–20 monosaccharide residues linked by glycosidic bonds that are readily hydrolyzed to their constituent monosaccharide’s either by acids (chemical) or by specific enzyme beta galactosidase (Prenosil et al 1987) (enzymatic). The enzymatic synthesis of GOS from lactose has been reported by many authors (Tzortzis, Goulas, Gibson, 2005; Onishi, & Tanaka1998; Wierzbicki & Kosikowski 1972; Cruz et al 1999). The concentrations and structures of the oligosaccharides produced are mainly dependent on the concentration of substrate (Boon et al, 2000). GOS stimulate the proliferation of lactic acid bacteria and bifidobacteria in the human intestine (Sako, Matsumoto, & Tanaka, 1999).

In this paper, a study on the enzymatic production of GOS from lactose by immobilization of whole cells of A. flavus was reported. The chemical synthesis of GOS requires multiple protection and de-protection steps (Sears & Wong, 2001). This complexity does not render this synthetic route attractive for industrial applications. To overcome this limitation, new strategies for the production of GOS by various methods of immobilizations were used (Aziz Tanriseven, S¸enay Dogan et al 2002). Purification of enzyme is expensive process, so it is desired to develop a new inexpensive method of production of GOS by using whole cells. The crude enzyme also performed well (Barbara Splechtna et al 2006) for the production of GOS to avoid expensive purification process. (Tzortzis, Goulas & Gibson, 2005) Whole cells of a novel strain, Bifidobacterium bifidum was used for the production of GOS.

Marine derived fungus A. flavus which is proven for the production of b-galactosidase, therefore it of interest to find whether the fungus A. flavus can produce GOS comparable to the GOS produced by the pure enzyme immobilization. In this study we try to find out the optimum conditions for the production of GOS from lactose by mutated cells of A. flavus.

Mechanism of action

In the enzymatic hydrolysis of lactose, the enzyme b-galactosidase transfers a galactose moiety to an acceptor containing a hydroxyl group. If this acceptor is a water molecule, galactose is liberated (hydrolysis). When a sugar moiety acts as the acceptor, galactosyl-oligosaccharides (GOS) are formed. (Fig.1) In addition to hydrolysis, b-galactosidase from various micro organisms is also known to catalyze trans-galactolysation reactions. The formed galactosyl-oligosaccharides vary in chain length and in the inter connection of the monomer units (Crittenden & Playne1996).

GOS are usually considered as non-digestible, mildly sweet, poorly viscous, water-soluble sugars. They are functional food ingredients with a great potential to improve the quality of many foods. GOS have various physiological functions, such as the improvement of the intestinal microflora based on the selective proliferation of Bifidobacteria and the stimulation of mineral absorption.

Flow diagram for the production of Galacto-oligosaccharides

Materials and methods

Chemicals

Lactose monohydrate was supplied from Merck; 2-nitrophenyl- β-D-galactopyranoside was procured from Carbosynth Limited, Berkshire, U.K. All other chemicals and medium constituents in this study were of analytical grade and procured from Sigma-Aldrich and s.d fine chemicals.

Microorganism

The Aspergillus flavus derived from marine fungus collected at Bay of Bengal near Vishakapatnam in  August 2005, using dilution-plate method (Christensen 1963) on PDA medium (Nielsen & Sorensen 1997).  It was identified according to its morphological characteristics and confirmed by Prof. Ananthpadnabhan (IMTECH, Chandigarh). Working stocks were prepared on Potato Dextrose Agar slants and stored at 4⁰ C. A. flavus was immobilized in calcium alginate and used for the production of GOS.

Enzymatic synthesis of GOS

Lactose solutions were prepared in different concentrations ranging from 0.5 M to 2.25 M in 50 mM potassium phosphate buffer containing 1 mM MgCl₂. Different reaction conditions were assayed: temperature (25⁰, 37⁰, 40⁰, and 50 ⁰C), pH (4.5, 5.5, 6.5, 7.0, 7.5, 8.0), Immobilized  Aspergillus flavus cells 2%. Experiments were carried out in duplicate. Reactions were performed in EM flasks incubated in an orbital shaker at 200 rpm. Samples were withdrawn at specific time intervals (30, 60, 120, 180, 240, and 300 min) and immediately immersed in boiling water for 5 min to inactivate the enzyme. The samples were stored at 4⁰ C for subsequent analysis.

Chromatographic determination of carbohydrates

The samples were analyzed by HPLC using amino column (Shodex Asahipak NH2P-50 4E) at room temperature. The column was eluted with acetonitrile : water (80 : 20)  at a flow rate of 1 ml/min. The peaks were detected by refractive index detector and it was assumed that the response was independent on the degree of polymerization. The amount of each saccharide was calculated from the area of corresponding peak. The number of sugar units of the oligosaccharides was found on the basis of retention time, trisaccharides and tetrasaccharides were identified on the basis of having a retention time later than of disaccharides. Lactose, glucose, galactose, and higher saccharides, such as tri- and tetrasaccharides were measured as weight percentage.

Samples and standard solutions of glucose, galactose, and lactose were filtered through a Millipore (0.45 micron) membrane before injection in to HPLC column. Twenty micro liters were injected using an auto sampler and separations were performed at a flow rate of 1 ml/min. Quantification of each sugar was performed by external calibration using standard solutions of galactose, glucose, lactose. The yield of GOS was expressed as percentage of carbohydrate weight formed per weight of lactose initially present. The peaks were measured with refractive index detector with the sample retention time 30 minutes.

Fig1.0. HPLC chromatogram:  carbohydrate profile obtained from lactose hydrolysis produced by immobilization of A. flavus at pH 7.00, 40⁰ C, 2.25 M of lactose solution. The identified compounds are indicated: (1) galactose, (2) glucose, (3) lactose, and (4, 5 and 6) Galacto oligo saccharides.

Results and discussions

Permeabilization of A. flavus with different chemical agents

Major part of enzyme β-galactosidase in cells of A. flavus was intracellular. So treatment with different chemical agent leads to permeabilization of cell wall there by the enzyme β-galactosidase, accessible with the substrate. Among various chemical agents (Fig 1.1) ethanol and toluene were found to be best permeabilizing agents for A. flavus followed by acetone.

Figure 1: Enzymatic reaction of lactose to Galacto oligosaccharides and glucose.   Click here to View figure

Permeabilization with Detergents

Among different detergents of cationic, nonionic, anionic were tried. (Fig 1.2) Out of which Cetyltrimethyl ammonium bromide shows best permeabilizing activity of the mutated cells of A. flavus. Various concentrations of CTAB ranging from 1.37×10^-3 M to 1.09×10^-2 M concentrations were tried. The cells of A. flavus after treatment with CTAB of 2.75×10^-3 M concentration (Fig 1.3) yields better β-galactosidase activity compare with the other organic solvents and detergents.

Figure 2: Flow diagram for the production of Galacto-oligosaccharides.   Click here to View figure

Figure 3   Click here to View figure

Effect of substrate concentration on production of GOS

Mutated and permeabilized cells of A. flavus were immobilized in sodium alginate and used for the production of GOS. For initial lactose concentrations of 0.5M, 0.75M, 1M,  1.25M, 1.5M, 1.75M, 2M and 2.25M were tried and the maximum GOS yields at 37⁰ C were found to be 4.2 %, 6.4 %, 12.4 %, 16.7 %, 22.1%, 23.9 % after 160 minutes, respectively. Transgalactosylation significantly increased with the increased lactose concentrations (Fig 1.4).This is due to the fact that, at a low lactose concentration, hydrolysis is favoured since the amount of hydroxyl groups of carbohydrates is lower as compared to those of water, which act as acceptors of galactose.

Figure 1a   Click here to View figure

Figure 2b   Click here to View figure

Figure 3c   Click here to View figure

Figure 4d   Click here to View figure

Effect of pH on the production of GOS

We also investigated the effect of pH on the production of GOS by using wide range Mcilvaine buffers at pH 4.5 to 8.0 at lactose concentrations 1.75M. The decrease in the lactose concentration is rapid due to hydrolysis of lactose at pH 5.5, but the amount of GOS formed was higher at pH 7.00. (Table 1.0). β-galactosidase from either E. coli or S. cervacea reported that optimum pH for production of oligosaccharides were little higher than optimum pH for hydrolysis (Hubber et al 1976). Our findings are consistent with the reported data.

Reusability of immobilized A. flavus cells

Cells were reused up to 8 times for subsequent synthesis reactions with equal amounts of GOS being formed at analogous periods of time. From this point onwards, and always at the same reaction times, a slight decrease on the produced oligosaccharides was observed (compared with the initial eight reactions), resulting to a total 10% decrease in the synthesis of oligosaccharides after 12 times of re-use.

Table 1: Effect of pH on the production of GOS.

Lactose solution (1.75 M) at different pH was incubated at 37⁰ C with mutated cells of A. flavus at a concentration of 2%  for 360 minutes.

Our results show that immobilization of whole cells was cost effective for the production of GOS. But the yield of GOS was comparatively low, with the immobilization of A.flavus. Immobilization of semi purified enzyme or crude extract also performed well in the production of GOS from lactose (Barbara Splechtna et al 2007). Degree of lactose conversion to GOS, mainly depend on the initial lactose concentration, the buffer (pH), salt concentration, temperature.

Conclusion

Based on the results reported in this study, β-galactosidase from A. flavus can be considered for the GOS production. To reduce enzyme costs, we successfully used the permeabilized A. flavus cells for the production GOS, thereby avoiding expensive purification of the enzyme. Moreover, the A. flavus cells can survive at 40⁰ C in immobilized form causes, improved yield of GOS and decrease in microbial contamination.

Acknowledgements

The author is very thankful to Dr R.S. Prakasham, Indian Institute of Chemical Technology Hyderabad for providing necessary facilities for analysis of Galacto oligosaccharides and his valuable suggestions.

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