LEVAN PRODUCTION BY BACILLUS SUBTILIS DSM 347 STRAIN

Artur Szwengiel, Maria Czarnecka, Halina Roszyk, Zbigniew Czarnecki

ABSTRACT

The tested strain of Bacillus subtilis DSM 347 was employed in investigations determining the impact of temperature and saccharose concentrations on the in vivo yield of levan synthesis. The best results were obtained in the treatment at 15% saccharose supplementation at 37°C. The obtained levan was subjected to acid and enzymatic hydrolysis and the composition of hydrolysates was examined using the spectrophotometric and densitometric methods on TLC plates.

Key words: Bacillus subtilis, levan, levansucrase..

INTRODUCTION

Levan is a homopolymer made up of fructose sub-units connected by b-2,6-glycoside and b-2,1-glicoside bonds in their branches. It develops in the course of a trans-glycosylation reaction with the participation of the levansucrase (EC 2.4.1.10) [7]. This fructan is manufactured, among others, by such microorganisms as: Zymomonas mobilis [15], Bacillus subtilis [4], Bacillus circulans, Bacillus polmyxa, Erwninia amylowora, Erwinia herbicola, Seraria sp. [18], Rahnella aquatilis [10], Pseudomonas syringae [8], Acetobacter xylium NCl 1005 [20].

There are many possibilities of utilising levan in the area of food, pharmaceutical and cosmetics production [17]. Moreover, levan can also be used as an emulsifier, formulation aid, stabilising thickener, surface-finishing agent, encapsulating agent as well as a carrier for colours and flavours in the food industry [9].

Levan is a non-digestible food fibre. Although it does undergo a partial de-polymerisation under the influence of the stomach juice, it is not digested by the pancreas secretion or small intestine juices [22] and, therefore, it can be treated as a component of prebiotic nature. In vitro investigations showed that bifidobacteria could utilise levan as a source of carbon. However, this depends on the degree of its polymerisation and as the upper limit, the various researchers suggest the molecular weight of approximately 4500 Da [13].

Levan can also be utilised to manufacture DFA lV (di-D-fructose-2,6’:6.2’-dianhydride). DFA lV belongs to the group of di-fructose anhydride compounds (DFAs) developed in the result of an intramolecular transfructosylation reaction. DFA lV is characterised by half the sweetness of saccharose, cyclical structure which guarantees its stability and, therefore, similarly to other DFAs, it can find application as a sweetener for diabetics [1].

At the present time, there are many polysaccharides on the market – primarily of plant origin – which play various functions in the food industry as well as in other industries. Nevertheless, attempts are made to find polymers, which apart from stabilising, thickening and other properties, could also fulfil other functional aspects in food products. Oligo-saccharides of prebiotic properties and polysaccharides with the structure, which can qualify them as components of food cellulose belong to glyco-biotechnological products. Since levan possesses many properties favourable and beneficial for human health, an attempt was made to synthesise this polymer employing, for this purpose, the strain of Bacillus subtilis DSM 347.

MATERIALS AND METHODS

The strain and media

The object of the performed investigations were bacteria of the Bacillus subtilis DSM 347 strain obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH. The strain was stored at the temperature of 4°C on slants with the following media composition (g/l): 1 – beef extract, 2 – yeast extract, 5 – pepton, 5 – NaCl and 15 – agar. The pH was brought to 7.4. The experimental strain was transferred regularly onto fresh slants and incubated at 28°C for 48 h.

The following growing substrate was used (g/l) during incubation: 100 (or 150, 200 and 250 – depending on the treatment) saccharose, 11.7 – citric acid, 4.0 – sodium sulfate, 4.2 – ammonium hydrogen phosphate, 5.0 – yeast extract and 100 ml/l salt solution (of the following composition (g/l): 4.62 – KCl, 4.18 – MgCl₂, 5.43 – MnCl₂, 0.49 – FeCl₃ and 0.21 – ZnCl₂). The substrate pH was brought to 6.8 by adding 25% ammonia water. All media were autoclaved at the temperature of 121°C for 20 min before culturing.

Culturing conditions

The growing medium was poured into Erlenmeyer’s flasks in the amount of 50 ml/flask. The inoculum was prepared by inoculating the growing medium with the pure culture from the slant (bacteria were washed by sterile, deionised water) and then shaken cultures were carried out at the temperature of 32 or 37° C for 16 h at 150 rpm. The inoculum (»2*10⁸ cells/ml) prepared in this way was used to inoculate the growing medium in flasks (10% in relation to the medium). The culturing process was carried out in aerobic conditions for 96 h. Samples were collected regularly every 12 h.

Determination of cell numbers

The cell count in 1 ml of the inoculum and the examined samples were determined with the assistance of the Thom chamber [11]. These measurements were used as the basis for the evaluation of the population growth in time.

Levan precipitation

Flasks with samples collected every 12 h were centrifuged at 4°C (10000 rpm, 20 min) [3]. The supernatant was then decanted and next 4 volumes of chilled 95% ethanol was added to it in relation to 1 sample volume [21], mixed and left for the night at 4°C. The liquid from the precipitated sediment was decanted and centrifuged with the aim to recover the low-density fraction suspended in it (10000 rpm, 20 min), the precipitates were mixed together and solved in deionised water and then heated in a boiling water bath for 10 min in order to deactivate exo-enzymes, cooled down and the levan was precipitated again. The obtained light-cream precipitate was dried at 60° C and stored at the desiccator.

Sugar content determination in levan

The total content of reducing sugars was determined in the reaction with DNS (λ = 530 nm) [14]. Fructose was determined on the basis of the Roe’e resorcin method, the measurement was carried out at the wavelength λ = 520 nm [19]. The glucose content was determined using the test containing glucose oxidase and peroxidase (λ = 510 nm) (Megazyme International Ireland Ltd. Ireland).

Protein determination in levan

Protein in the levan was determined using the method of Lowry [12].

Yield determination

The quantity of the levan was estimated using the gravimetric method (g of levan/l of growing medium). The yield was calculated in relation to saccharose and in relation to the introduced fructose residues with saccharose.

Mild acid and enzymatic hydrolysis of levan

Levan was subjected to a mild acid hydrolysis in the 0.01N HCl environment and to the enzymatic hydrolysis (inulinase EC 3.2.1.7 derived from Aspergilus niger). The course of the levan hydrolysis was monitored in time with the assistance of:

1. measurements of the medium reduction changes in the reaction with DNS,

2. the use of the planar chromatography.

Spot visualisation was performed employing the method of Roe and Papadopoulos [19] as modified by Muro et al. [15] (on Silica gel 60 TLC plates from Merck, Catalogue no. 1.05626) in conjunction with the densitometric measurement (Vitatron TLD 100).

RESULTS AND DISCUSSION

The initial cell-count in 1 ml inoculum of bacteria amounted to »2*10⁸. During the 96 h of culturing, an increase in the bacterial population in 1 ml of the culturing liquid media was observed (from the initial value of »7*10⁷ to »9*10⁹ cells/1 ml). The size of the examined population was lower at the temperature of 32°C, in comparison with the temperature of 37°C. The microscopic examinations revealed that the cells from the lower temperature were less mobile and bigger. During the first 12 h of culturing, the pH of the culturing medium decreased from 6.80 to about 6.10, only to reach the value of over 7.00 at the termination. The observed correlation can be attributed to the fact that during the vegetative growth, bacteria gather organic acids, whereas towards the end of the exponential growth, when easily available carbon sources begin to run out, cells begin to get ready to produce endospores. Krebs cycle enzymes become unblocked and the acetyl-CoA undergoes complete oxidation to CO₂. Then, in aerobic conditions, the accumulated organic acids become oxidised and the pH of environment increases [11].

On the basis of experimental data showing changes in the levan content in time in various culturing treatments (Fig. 1), mathematical models were developed (Table 1). The models allowed to determine the points corresponding with maximum values of the levan (g/l) precipitated in the examined time interval. The determined points were used to calculate in vivo yields of levan synthesis in relation to the saccharose, which is the donor of fructose residues as well as to fructose itself (Fig. 2). The best results in the conversion of saccharose to levan were achieved in the 33^rd hour (Fig. 1 and Fig. 2). The medium containing 15% (w/v) saccharose and the process was carried out at 37°C. The obtained 36% yield in relation to the introduced saccharose proved that approximately three fourths of the fructose residues introduced into the medium were incorporated into the develop ed polymer. Investigations on the precipitation of inulin from aqueous solutions with the assistance of 95% ethanol confirmed that molecules of low degree of polymerisation (DP 1-10) remained in the solution [23], which suggests that the method of levan separation with the assistance of ethanol is loaded with an error because it does not allow precipitating molecules of low degree of polymerisation.

Table 1. List of functional correlation coefficients on the basis of which maximum levan yields were determined in the examined time interval in relation to culture treatment (Gauss model ; rational function

In each of the analysed cases, the yield of levan synthesis at 37°C was higher than at 32°C. In addition, the time during which the highest quantities of levan were precipitated was also considerably shorter at the higher temperature. However, it is difficult, on the basis of the performed experiments, to conclude definitely if the increased temperature exerted a significant influence on the secretion of the levansucrase into the medium or if it was caused by the change of the kinetics of the enzyme itself. Despite obtaining very similar yields on media with 10 and 15% (w/v) saccharose concentrations (Fig. 2), the treatment with the temperature of 37°C and 15% (w/v) supplementation was selected as the most favourable, primarily because of the higher weight of the obtained polymer from one unit of volume. A similar yield was reported in investigations conducted by Euzenat et al. [5] who worked on the determination of the effect of the saccharose concentration and temperature on the levan synthesis on the basis of the enzyme obtained from Bacillus subtilis C4 media. Each of the presented curves on Figure 1 has its extreme in the examined time interval. There are several ways the different runs of these curves can be explained. One of them assumes that part of the highly polymerised levan, or the levan attached to the surface of bacterial cell walls underwent centrifugation in the course of the removal from the biomass post-culturing liquid. In addition, it can also be presumed that once the sources of the easily available carbon (i.e. glucose, which initially, resulted in a significant increase of medium reductivness; unpublished results) had been depleted, bacteria began to utilise also fructose, which constituted the basic building blocks of levan.

The total content of reducing sugars, glucose and fructose were also determined in the precipitated levan. Results of these analyses are presented in Figure 3. It is probable that part of the determined protein remains in the active enzyme – levan complex [6], whereas another part comprises proteins which underwent precipitation with the supernatant in the result of high ethanol concentration.

Acting on the assumption that each levan molecule has a glucose molecule at one of its ends [2], it can be assumed on the basis the analysis of the carbohydrate composition of the obtained levan (Fig. 3) that the degree of fructan polymerisation amounted, on average, to 20 fructosan sub-units.

Levan samples obtained after 33 h of culturing at the temperature of 37°C on the substrate containing 15% (w/v) saccharose were subjected to 0.01N HCl hydrolysis as well as to enzymatic hydrolysis with the aim to test the susceptibility of the obtained product to the conditions of mild acid hydrolysis as well as to the action of inulinase. The performed densitometric analysis (the released fructose was subjected to detection) of the levan mild acid hydrolysis products confirmed the possibility of obtaining fructo-oligosaccharides (FOS) in this way, whereas the hydrolysis employing inulinase resulted in the development of only free fructose (Fig. 4, diagrams A and B). Since the employed method for fructose detection allowed visualisation of only those spots which contained fructose, Figure 4 presents also those curves which portray the increase of reductiveness of the reaction mixture using, for this purpose, the reaction with DNS. The small differ ences between the employed spectrophotometric determination of the content of reducing sugars and the densitometric measurement of the fructose liberated during the 0.01N HCl hydrolysis proved that fructose was the principal product of hydrolysis. Results of the applied linear regression analysis (Fig. 5) between values obtained in the result of determination of the content of reducing sugars in the reaction with DNS and the content of fructose determined using the densitometric method indicated that in the described system, the spectrophotometric determination can easily substitute the densitometric measurement (directional coefficients of the linear equation in both cases are close to ‘1’ and the high determination coefficient (R²) confirms high correlation of the two methods).

The above-discussed investigations constitute an initial analysis of the impact of temperature and substrate concentration on the in vivo levan formation in relation to the tested strain. The performed experiment allowed concluding that the best conversion yield of the saccharose into levan using the Bacillus subtilis DSM 347 strain in the described experimental conditions were achieved using a 15% (w/v) addition of saccharose and culturing temperature of 37°C. On the other hand, subjecting the obtained levan to mild acid hydrolysis, it is possible to obtain a mixture of short-chained FOS and fructose. In addition, levan undergoes enzymatic hydrolysis to free fructose using inulinase.

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