GB2065689A - Enzymatic clarification of polysaccharide solutions - Google Patents

Abstract

Acid and/or neutral proteolytic enzymes are used for the degradation of the cells of the genus Xanthomonas as well as of those of other polysaccharide-producing organisms in order to clarify xanthan gum or other polysaccharide. The protease may be elaborated by Bacillus subtilis or Aspergillus niger.

Description

SPECIFICATION Enzymatic clarification of polysaccharide solutions The present invention relates to the clarification of solutions of polysaccharides, and in particular to the use of enzymes to effect such clarification.

The presence of non-viable microbial cells in polysaccharide fermentation broths and crude polysaccharide preparations isolated from whole broths can result in adverse effects when the preparations are to be used in applications such as food formulation or enhanced oil recovery.

Several chemical and physical methods for removing cells have been proposed; on the whole these known methods either result in chemical degradation or modification of the polysaccharide or are costly.

UK Patent Specification No. 1443507 teaches that only a certain type of enzyme, the bacterial alkaline proteases, can be used to degrade bacterial cells of the polysaccharide producers of the genus Xanthomonas. The literature references cited in the specification indicate that the alkaline proteases used are characterized by their high proteolytic activity above pH 7, the presence of an esterase activity and the presence of a serine moiety at the active site which is inactivated by diisopropyl fluorophosphate.

We have now found it is possible to use other enzymes for the degradation of the cells of the genus Xanthomonas as well as of those of other polysaccharide-producing organisms.

Our invention employs acid and/or neutral proteolytic enzymes, and has the advantage that one can avoid working at an alkaline pH which is required for optimum activity of the alkaline proteases and which is often detrimental the polysaccharide being treated. Moreover, the present invention is surprising in view of our previous finding that when employed under different conditions and at different concentrations, the proteases do not affect the viability of bacteria of the species Azotabacter vinelandii.

In accordance with the present invention there is provided a method for the clarification of a polysaccharide solution containing bacterial cells in which method the cells are degraded using an acid and/or neutral protease.

The enzymes employed in the present method include acid and neutral proteases from bacterial, fungal or plant sources and contain either a sulphydryl, metal ion or carboxyl group at their active site (A. Yamamoto, Proteolytic Enzymes in "Enzymes in Food Processing", Ed. G. Reed. 1975, Academic Press).

Thus whereas the process known for UK Patent Specification No. 1443507 employs enzymes with a serine moiety at the active site, the present method employs enzymes which have a different functionality at the active site. This difference in functionality is in turn reflected by the different proteolytic characteristics which are used to advantage in the present method.

The acid proteases are normally most active in the pH range of 2 to 5, and are insensitive to certain reagents which inhibit alkaline proteases. The neutral proteases are normally most active at around pH 7.

In contrast, the alkaline proteases exhibit maximum activity at a pH of 9.5 to 10.5.

As the present invention employs acid or neutral proteases, it is now possible to work at a nonalkaline pH and yet obtain maximum activity from the enzyme being used.

Proteases for use in the present method are readily available.

Such proteases include for example the neutral proteases elaborated by Bacillus subtilis and the acid proteases elaborated by Aspergillus niger or present in pineapples.

When using neutral proteases the pH of the solution being treated is suitably from 4 to 9, preferably between 6 and 7, while for acid proteases the pH is suitably from 2 to 7, preferably from 3 to 6.

The level of protease employed will depend on the nature of the solution being treated and on the conditions to be employed in effecting the clarification. For instance, with solutions of xanthan gum (the polysaccharide produced by Xanthomonas spp) a concentration of 0.2 to 4 gram of enzyme per litre of solution will usually be found to give satisfactory clarification. On the other hand, with solutions of microbial alginate (produced byAzotobacter vinelandii) a concentration of 0.5 to 5 gram of enzyme per litre of solution will usually be appropriate.

Generally, it is preferred that sufficient enzyme is added to give an enzyme activity in the solution being treated of 2 to 10 Ansons per litre, for instance 4 to 8 Ansons per litre (the Anson being that amount of enzyme which will release 1 milliequivalent of tyrosine per minute from denatured haemoglobin at 300C and pH 7.5).

In performing the present method, the degradation of the bacterial cells is achieved by incubating the enzyme-containing solution for a suitable period, for example from 1 to 10 hours, and preferably at above room temperature. Preferred incubation conditions are 1 to 8 hours at 40 to 550C.

The present treatment can be effected on a batch or continuous basis, with continuous operation being particularly suited for large-scale industrial use.

As a result of the cell degradation, a solution with greater clarity is obtained. Moreover, the polysaccharide purity is often increased.

The present invention is illustrated by the following non-limiting examples.

EXAMPLE 1 400 g of a xanthan gum fermentation broth was adjusted to pH 6.5 with hydrochloric acid and warmed to 5O0C. The neutral protease known as 'Neutrase' (Trade Mark, Novo Industries) and obtained from Bacillus subtilis was added to the broth to a final concentration of 0.25% (w/v). Stirring was applied to produce a homogeneous broth which was then left in a water bath at 500C for 7 hours.

400 g of untreated broth serving as a control was similarly left for 7 hours.

At the end of the 7 hours the clarity of the broth was assessed using an EEL colorimeter. The particle size reduction of a 1 :100 dilution of the broth in distilled water was assessed by measuring optical densities at 540 nm using a Perkin Elmer 6/20 spectrophotometer. The greater the reduction in adsorption after filtration through a 0.45 micron millipore filter using an AP200 glass fibre pre-filter, the lower the degree of particle size reduction which has occurred. The results are shown in Table 1: TABLE 1 Control Enzyme-treated Colorimeter Reading 10 7 Optical Density at 540 nm (a) Pre-filtration 0.13 0.03 (b) Post-filtration 0.0 0.03 The polysaccharide was precipitated from the broth by the addition of 2 volumes of isopropyl alcohol.The precipitate was then freeze-dried and milled. 0.1% solutions of the treated and untreated (control) dried and milled polysaccharide were prepared in distilled water and the absorption at 540 nm determined. The filterability of 0.1% solutions of the polysaccharide in 3% sodium chloride was assessed by measuring the filtration rate after passage of 1 50 g of the solution through a 0.8 micron millipore filter and an AP200 millipore pre-filter at 20 psi and room temperature. The results are shown n Table 2: TABLE 2 Control Enzyme-treated Optical Density at 540 nm 0.113 0.01 Filtration rate (g sec-') 0.003 0.12 after 150 gms.

EXAMPLE 2 400 g of a xanthan gum fermentation broth was adjusted to pH 3 with HCI and warmed to 500C using a water bath. 0.25% of 'Acid Fungal Protease' produced by the company Miles from Aspergillus niger was added. The broth was left for 3.5 hours, neutralised with NaOH and the polysaccharide recovered as in Example 1. The filterability of 0. 19/0 solutions in distilled water and 3% NaCI was assessed as described in Example 1. A control sample was prepared and assessed as in Example 1. The results obtained are given in Table 3: TABLE 3 Control Enzyme-treated Water 3% NaCI Water 3% NaCI Filtration rate (g sec-1) 0.35 0.003 10 10 after 150 g EXAMPLE 3 400 g of a xanthan gum qas adjusted to pH 6.8 with HCI and warmed to 500C using a water bath.

To this was added 0.25% of the neutral protease 'H.T.Proteolytic 200' Miles, produced by Bacillus subtllis. The broth was then left for 7 hours along with a control without enzyme. The clarity of both broths was assessed at the end of this period, as in Example 1. The polymer was precipitated from the broths, dried and a 0.1% solution in distilled water measured for optical density at 540 nm, as in Example 1. The results are shown in Table 4: TABLE 4 Control Enzyme-treated Colorimeter reading 10 6.1 Optical Density at 540 nm 0.113 0.007 EXAMPLE 4 400 g of a xanthan gum broth was adjusted to pH 5.8 with HCI and warmed to 500C using a water bath. To the broth was then added 0.25% of the acid protease 'Bromolain' (Miles, isolated from pineapples). The broth was left for 7 hours along with a non-enzyme-treated control.The optical density of the 0.19/0 diluted broth, pre and post-filtration was measured as described in Example 1. The polysaccharide was recovered by precipitation with isopropyl alcohol, dried, milled and a 0.1% solution in distilled water and 3% NaCI assessed for filterability, as described in Example 1. The results obtained are shown in Table 5: TABLE 5 Control Enzyme-treated Water 3% NaCI Water 3% NaCI Filtration rate (g sec-1) 0.35 0.003 60 2.2 after 150 9 Optical density at 540 nm (a) pre-filtration 0.13 0.035 (b) post-filtration 0.0 0.035 EXAMPLE 5 400 g of a xanthan gum broth was adjusted to pH 6.5 with HCI and heated to 500C with a water bath. To the broth was added 0.25% of the neutral protease 'Bacterial Protease' (ABM Chemicals, produced by Bacillus subtilis).The broth was left for 4 hours along with a non-enzyme-treated control.

The polysaccharide was precipitated from the broth using isopropyl alcohol, then dried and milled. 0.19/0 solutions of the treated and untreated products in distilled water were assessed for optical density at 540 nm, as described in Example 1. The results are shown in Table 6: TABLE 6 Control Enzyme-treated Optical Density at 540 nm 0.113 0.02 EXAMPLE 6 The effect of enzyme treatment of viscosity was determined by preparing a 1% solution,s.of the enzyme-treated products in distilled water and comparing the viscosity with a 1% solution of the control product. The enzyme treated products were prepared by the techniques outlined in Examples 1 to 5.

Viscosities were determined using a Rheomat 30 viscometer with cone-and-plate attachment and are shown in Table 7 as the apparent viscosity at a shear rate of 1 set'.

TABLE 7 Viscosity(cp) at 1 sec-1 Control Enzyme-treated 1. Acid Fungal Protease 5,600 5,500 2. H T Proteolytic 200 5,600 5,500 3. Bacterial Protease 4,000 4,200 It can be seen that the enzyme treatments did not adversely affect the viscosities.

EXAMPLE 7 600 kg of a xanthan gum broth containing 2.7% by weight of the gum was heat-treated at 1200C + 20C for 2 minutes in order to effect an initial improvement of the rheology and other properties of the gum. After adjusting the temperature of the broth to 470C. sufficient 'Neutrase' solution was added to give an enzyme concentration in the broth of about 1.7 g/l. The mixture was held at 470C for 2 hours, and then the enzyme was inactivated by raising the temperature to 900C for 2 minutes. The xanthan gum was then precipitated from the broth using isopropanol, and dried.

Measurement of the optical density at various stages gave the results shown in Table 8.

TABLE 8 Optical Density Sample at 540 nm After heat treatment at 1200C, 2 minutes 4.0 After enzyme treatment at 470C, 2 hours 0.5 After enzyme inactivation at 900C, 2 minutes 0.8 Apart from the evident improvement in optical clarity, the enzyme treatment also led to an improvement in the purity of the product. Thus, the nitrogen content feli from 1.7% to 1.0% as a result of the enzyme treatment. Moreover, the decarboxylation assay increased favourably from 4.0% to 4.4%.

The enzyme treatment also led to beneficial effects on the rheology of the xanthan gum. The consistency index K (cp) and the flow behaviour index of the gum at 1% in 3% sodium chloride solution were 9,600 and 0.15, respectively, immediately before the enzyme treatment and were 17,400 and 0.10, respectively, immediately after the enzyme treatment.

EXAMPLE 8 The procedure of Example 7 was repeated on a continuous basis instead of a batch basis.

Using a flow rate of approximately 100 kg/hr, xanthan gum broth was heat-treated, at about 12500 for about 1.7 minutes, cooled to about 45 C, and transferred to a continuous enzyme incubator vessel of working capacity 180 litres. The enzyme was continuously added as a 10% solution to give a Neutrase level of about 1.5 g/l. Incubation was at 500C, about pH 6.8, for a mean residence time of about 1.9 hours. Following enzyme treatment the broth was heated to about 900C for a residence time of about 1.9 minutes, and then precipitation was effected using isopropanol.

By this continuous procedure, the xanthan gum was upgraded in the manner shown in the following Table 9.

TABLE 9 Sample Pre-treatment Post-precipitation Nitrogen (%) 1.85 1.04 Decarboxylation (%) 4.15 4.38 Sulphated Ash (%) 12.25 9.61 Consistency index (K, at 0.3% in 3% NaCI) 460 1050 Optical Density (540 nm) 4.76 1.10* * measured just before precipitation EXAMPLE 9 1000 ml of an alginate-containing culture broth of Azotobacter vinelandii obtained by a continuous culture in accordance with the process of UK Patent No. 1 51 3104 was adjusted to pH 6.5 and heated to 500C. 20 ml of a filtered solution of the enzyme preparation HT Proteolytic 200 (Miles Biochemicals, activity of 200 NUS/g) was added, with stirring to the broth. A control was set up in a similar manner using 20 ml distilled water instead of the enzyme solution.After incubation at 500C for 4 hours the products were precipitated by addition to 98% isopropyl alcohol and dried under vacuum at 400 C. The products obtained were dissolved in distilled water to a concentration of 0.1%. The degree of turbidity was measured using optical density measurements as described in Example 1. The results are shown in Table 10.

TABLE 10 Control Enzyme-treated Optical Density at 540 nm 0.20 0.05 EXAMPLE 10 1000 ml of an alginate-containing culture broth of Azotobacter vinelandii obtained as in Example 9 was adjusted to pH 6.5 using 2M HCI and heated to 500C. A 100 gl-' solution of the enzyme preparation Neutrase (Novo Industries Ltd, activity of 3.0 Anson units/g) was filtered to remove the inert carrier and 20 ml of the filtrate added, with stirring, to the broth. A control was set up in a similar manner having 20 ml distilled water added instead of the enzyme solution. Ater incubation at 500C for 4 hours the products were precipitated by addition to 98% isopropyl alcohol and dried at 450C under vacuum. The products obtained were dissolved in distilled water to a concentration of 0.1%. The turbidity of the solutions were estimated as before. The results are given in Table 11.

TABLE 11 Control Enzyme-treated Optical Density at 540 nm 0.2 0.055

Claims (14)

1. A method for the clarification of a polysaccharide solution containing bacterial cells in which method the cells are degraded using an acid and/or a neutral protease.

2. A method according to claim 1 which employs a neutral protease at a pH of from 4 to 9.

3. A method according to claim 1 or 2 which employs a neutral protease elaborated by Bacillus subtilis.

4. A method according to claim 1 which employs an acid protease at a pH of from 2 to 7.

5. A method according to claim 1 or 4 which employs an acid protease elaborated by Asperglllus niger or present in pineapples.

6. A method according to any preceding claim when used for clarification of a xanthan gum solution.

7. A method according to claim 7 which employs a protease concentration of 0.2 to 4 gram per litre.

8. A method according to any of claims 1 to 5 when used for clarification of a solution of microbial alginate.

9. A method according to claim 8 which employs a protease concentration of 0.5 to 5 gram per litre.

10. A method according to any preceding claim, wherein the protease is added at a level giving an enzyme activity of 2 to 10 Ansons per litre of polysaccharide solution.

11. A method according to any preceding claim in which the degradation is effected for 1 to 8hoursat40to550C.

12. A method according to any preceding claim, when performed as a continuous process.

13. A method for continuous clarification of a xanthan gum-containing broth produced by continuous fermentation of Xanthomonas sp, in which method an acid and/or a neutral protease is continuously added to the broth and a xanthan gum of better purity as well as better clarity is continuously obtained.

14. A method according to claim 1 and substantially as described in any of the Examples herein.