Xanthan production from whey treated with immobilized lactase

MATERIALS AND METHODS

Bacterial strains: Streptococcus salivarius subsp. thermophilus EMCC 10509 was obtained from the microbial culture collection center of Cairo MIRCEN, Fac. Agric., Ain Shams Univ., Egypt and maintained on YPG medium which containing (g/L); yeast extract, 5.0; peptone, 10.0; glucose, 5.0; NaCI, 5.0 and agar 20.0. The pH was adjusted to 7.2-7.4. Xanthomonas campestris NRRL B-1459 was used throughout this study. The strain was maintained on slants with YPGM medium (Rajeshwani et al., 1995).

Whey Preparation: Whey from bovine was supplied as sweet dried whey from Sigma Co., USA. It contained (w/w) 73 percent lactose, 10.8 percent protein and 0.75 percent phosphorus. Deproteinized whey solutions were prepared by acidification (pH 4.5) and heat treatment (90°C for 30 min). Then, the preparation was cooled and filtered to remove the precipitated protein.

Lactase Production: Lactase was produced in optimum conditions as described by Menshawy (1997). The medium employed for lactase production contained 5.48 percent sweet dried whey (Sigma Co.) in water together with "supplementary nutrients" [0.5 % of NaH₂PO₄; 0.5 percent of yeast extract, and 0.3 percent of (NH₄)₂SO₄1. The initial pH was adjusted to 7.0. The strain was grown in shake flasks (120 rev/min) for 24 h at 40°C.

Extraction and Purification of Lactase: Harvested cells were washed twice with 0.067 M potassium phosphate buffer, pH 6.8. Washed cells were suspended in 25 ml of 0.067 M potassium phosphate buffer, pH 6.8, containing 0.5 mM MgSO₄ and 0.1 mM MnCl₂. The suspension was treated with toluene (2 percent v/v) at 37°C for 10 min. Broken cells were removed and discarded by centrifugation at 15 000 rpm for 30 min at 4°C and the supernatant was used as an enzyme source. Cell free extract (1000 ml) was collected and precipitated by the addition of one volume of acetone at 4°C. Then, the precipitate was collected by centrifugation and the residual acetone was evaporated using an air stream. The precipitate was dissolved in phosphate buffer at pH 7.0 and dialyzed at 4°C against the same buffer. Solid (NH₄)₂SO₄ up to 60 percent saturation was added slowly to the dialyzed solution, the precipitate which formed was collected by centrifugation at 15000 rpm for 20 min in refrigerated centrifuge, resuspended in phosphate buffer, pH 7.0 and dialyzed extensively against the same buffer at 4°C. The partially purified enzyme was applied on a Sephadex G-100 column to remove the (NH₄)₂SO₄, then active fraction was collected and used as a source of lactase.

Preparation of Immobilized Lactase: Chitosan beads were prepared by shaking 0.5 g chitosan in 10 ml of 0.1 M HCI containing 2.5 percent glutaraldehyde for 2 h at 30°C. The beads were precipitated by the addition of 0.1 M NaOH to neutrality. The beads were collected by filtration and washed with water. The wet chitosan beads were mixed with 5.0 ml of the lactase solution (1000 units). After being shaken for 1 h at 30°C, the unbound lactase was removed by washing with distilled water. In case of chitin, 0.5 g chitin was shaked in 10 ml of 2.5 percent glutaraldehyde in 0.1 M acetate buffer (pH 5.5) for 2 h. The solid material was filtered and washed. The treated chitin was mixed with 5.0 ml of the lactase solution (1000 units) for 1 h at 30°C. The unbounded lactase was removed by washing with distilled water (Ohtakara, 1988).

Assay of Immobilized Lactase Activity: Lactase (approximately g of protein) were suspended in 50 mmol/l of phosphate buffer (pH 6.9) containing 4.56 percent whey lactose at 40°C with magnetic stirring. After 10 min of incubation or at various times a portion of the reaction mixture was removed and the amount of lactose hydrolyzed was assayed. One unit of immobilized lactase was defined as the amount of immobilized lactase required to hydrolyze 1 μmmol of lactose per min at 40°C and pH 7.0.

Whey Hydrolysis with Immobilized lactase: Whey permeate, adjusted to pH 7.0, was hydrolyzed using immobilized lactase. Hydrolyzed whey permeate was supplemented with a basal medium (per liter K₂HPO₄, 7 g; KH₂PO₄, 2 g; NH₄NO₃, 0.6 g; FeSO₄.7H₂0, 0.01 g; MgC1₂.6H₂0, 0.1 g and MnC1₂, 0.001 g; Na-citrate, 2 g and 0.125 percent tryptone). The pH of the solution was finally adjusted to 7.0 with 2N H₂SO₄ and 2N KOH. 50 ml of whey medium was dispensed into 250 ml Erlenmeyer flasks, sterilized, then, 5 ml of 24 h old inoculum was added (for preparing inocula, a fresh culture grown on the YPGM agar slant at 28°C for 48 h was transferred to 500 ml Erlenmeyer flasks containing 100 ml of the YPGM broth and incubated at 28°C and 250 rec/min for 24 h).

Xanthan Production: In shake-flask (250 rev/min on a rotary shaker) experiments, cultures were grown at 30°C using 250-ml Erlenmeyer flasks containing 50 ml of liquid xanthan production medium (a basal medium amended with different sugar sources) and inoculated by 10 percent (v/v) inoculum. Inocula was prepared as previously mentioned.

Sugar determination: Glucose was determined by glucose oxidase peroxidase. Galactose was determined by galactose oxidase peroxidase. Lactose was determined by Nickerson et al. (1976) method.

Xanthan Determination: Cultures were centrifuged at 10,000 xg for 10 min. To aliquots of the supernatant ethanol was added to 70 percent concentration with agitation followed by standing in cold for 3 hr. The precipitates were collected by centrifugation and redissolved in aliquots of distilled water. Modified Anthrone method (Trevelyan and Harrison, 1952) was used for quantitative determination. The standard curve was constructed by using pure xanthan.

Viscosity Measurement: Viscosity was measured using a rotational viscometer at a constant shear rate of 10.71S^–1 and 30°C.

pH Determination: The pH value was measured using laboratory pH-meter with glass electrodes (Knick-Digital-pH meter 646).

Protein determination: Protein was determined by the method of Lowry et al. (1951), using crystalline bovine serum albumin as standard.

Lactase activity determination: The activity of lactase was determined at 35°C using 45 mM lactose as substrate (in 50 mM phosphate buffer, pH 7.0). Glucose evolution was analyzed using the glucose oxidase/peroxidase system (Bergmeyer and Bernt, 1974). One unit of lactase activity is defined as the amount of enzyme that hydrolyses 1 mmmol lactose/min at 35°C.

RESULTS AND DISCUSSION

Purification of lactase: Purification parameters are summarized in Table 1. A 29.25-fold purification of the lactase was achieved with an overall recovery of 59.29 percent.

Table 1:	Summary of the partial purification of lactase from S. salivarius subsp. thermophilus

Table 2:	Immobilization of lactase by covalent binding on chitosan and chitin

Table 3:	Lactose hydrolysis by lactase at 37°C. Experimental conditions: 5.48% deproteinized whey (4% lactose), pH 7.0, 15 U lactase/ml

Fig. 1:	Lactase production and lactose utilization during growth in different concentrations of whey.

The results are in line with Rao and Dutta (1981).

Immobilization of lactase: The immobilization of S. salivarius subsp. thermophilus lactase by covalent binding though glutaraldehyde onto chitin and chitosan was carried out (Table 2). A considerably good loading efficiency (354 units/g) for lactase immobilized on chitin, but low immobilization yield was detected (70 percent). Lactase immobilized on chitosan by covalent binding showed higher activity (425 units/g) and better immobilization yield (85%). Therefore, chitosan was used for lactase immobilization for the purpose of whey hydrolysis. Immobilization of lactase on chitosan might be useful in processing milk by-products into valuable sweeteners, or into nutritional media for fermentation processes. Its possible application to solving many problems related to the use of lactose should also be considered (Leuba and Widmer, 1977; Greenberg and Mahoney, 1981).

Whey lactose hydrolysis: Quite high activity for the hydrolysis of lactose was obtained by immobilized lactase. Table 3 is indicated the concentrations of glucose produced during hydrolysis of 5.48 percent whey (4.0 percent lactose) with the lactase immobilized onto chitosan. A maximum of 20 percent glucose of the total lactose was obtained by 71 percent conversion of lactose, which is slightly less than with the free lactase. The results are in line with Huffman and Harper (1985) and Mozaffar et al. (1986).

Fig. 2a:	X.campestris behavior in xanthan production medium amended with glucose. Sugar consumed (∇), Xanthan (●), viscosity (○), pH (▴)

Xanthan production: The basal medium was supplied with glucose, galactose, or both of them (mixture of glucose and galactose 50/50) and used as xanthan production medium by X. campestris. The results given in Fig. (2a-c) revealed that glucose and galactose either alone or in combination were utilized for xanthan synthesis. Glucose was consumed more rapidly than galactose. The results also showed simultaneous utilization of both sugars. After 72 h of batch fermentation, 17.22 (g/L) of xanthan accumulated in the broth. This maximum yield recorded 86 percent after 72 h by using glucose medium.

When lactose used in the production medium as a sole carbon source, slight amount of lactose was assimilated by the tested organism. Only 0.4 g of xanthan accumulated in the liter broth after 72 h of batch fermentation (Fig. 2d).

Fig. 2b:	X. campestris behavior in xanthan production medium amended with galactos. Sugar consumed (∇), Xanthan (●), viscosity (○), pH (▲)

Fig. 2c:	X. campestris behavior in xanthan production medium amended with glucose+galactose. Sugar consumed (∇), Xanthan (●), viscosity (○), pH (▲)

Fig. 2d:	X. campestris behavior in xanthan production medium amended with lactose. Sugar consumed (∇), Xanthan (●) viscosity (○), pH (▲)

Fig. 2e:	X. campestris behavior in xanthan production medium amended with whey. Sugar consumed (∇), Xanthan (●), viscosity (○), pH (▲)

Fig. 2f:	X. campestris behavior in xanthan production medium amended with pretreated whey. Sugar consumed (∇), Xanthan (●), viscosity (○), pH (▲)

The low level of lactase present in X. campestris may explain this behavior. It was expected according to Stauffer and Leeder (1978) who found that no significant amount of xanthan gum is produced when X. campestris is grown in lactose medium. Frank and Somkuti (1979), Walsh (1984) and Fu and Tseng (1990) reported that X. tampestris was not able to grow in a lactose medium and to synthesize high xanthan yield because of the low concentration of produced lactase. Moreover, Drahovska and Turna (1995) found that original strain X. campestris 1069 produced 10.5 g of xanthan gum in liter of glucose medium after 16 h and no detectable amount of xanthan in lactose medium.

Slight amounts of lactose from whey were assimilated by X. campestris in whey medium, the yield percent was very low (1.75 percent) and only 0.35 g xanthan /L after 72 h (Fig. 2e). This result in agreement of the previous data about xanthan production in lactose medium, therefore, an attempt to use lactose-based substrate (such as whey) for gum production by X. campestris would be difficult. Some attempts have been previously made by several groups to construct lactose utilizing X. campestris strains for xanthan production. Fu and Tseng (1990) were able to select a strain which can utilize lactose for xanthan production, but the strain was not stable.

Therefore in this study, a mobilizable lactase was introduced into whey firstly to hydrolyse lactose to fermentable sugar, glucose and galactose. In that case, the bacterium was able to grow and successfully produce xanthan from pretreated whey in amounts near to those produced by galactose-grown cells, reached to 12.99 g xanthan/L after 72 h (Fig. 2f) and was 40-fold higher than in a medium containing whey. The results obtained are an indication for the possibility usage of pretreated whey by immobilized lactase in the process of industrial production of xanthan.