AU2001252219A1

AU2001252219A1 - Beta-glucans from filamentous fungi

Info

Publication number: AU2001252219A1
Application number: AU2001252219A
Authority: AU
Inventors: Federico Federici; Maurizio Petruccioli; Laura Selbmann; Francesca Stingele; Peter Van Den Broek
Original assignee: Societe des Produits Nestle SA
Current assignee: Societe des Produits Nestle SA
Priority date: 2000-03-24
Filing date: 2001-03-20
Publication date: 2001-12-20
Anticipated expiration: 2021-03-20

Description

BETA-GLUCANS FROM FILAMENTOUS FUNGI

The present invention relates to a method of producing a beta-glucan; use of a non-pathogenic saprophytic filamentous fungus or composition comprising it for providing a beta-glucan and thereby improving food structure, texture, stability or a combination thereof; use of a non-pathogenic saprophytic filamentous fungus for providing a beta-glucan and thereby providing nutrition; and use of a fungus or composition comprising it in the manufacture of a medicament or nutritional composition for the prevention or treatment of an immune disorder, tumour or microbial infection.

Within the context of this specification the word "comprises" is taken to mean "includes, among other things". It is not intended to be construed as "consists of only".

Over the last decade there has been a great deal of interest in biopolymers from microbial origins in order to replace traditional plant- and animal derived gums in nutritional compositions. New biopolymers could lead to the development of materials with novel, desirable characteristics that could be more easily produced and purified. For this reason, characterisation of exopolysaccharide (EPS) production at a biochemical as well as at a genetic level has been studied. An advantage of EPS is that they can be secreted by food micro-organisms during fermentation, however, using EPS produced by micro-organisms gives rise to the problem that the level of production is very low (50-500 mg/1) and that once the EPS is extracted it loses its texturing properties.

One example of an EPS is a beta-glucan. Beta-glucans are made of a β- glucose which are linked by 1-3 or 1-6 bonds and have the following characteristics that are attractive to the food- industry: viscosifying, emulsifying, stabilising, cryoprotectant and immune-stimulating activities.

Remarkably, it has been found that fungi can produce high amounts of biopolymers (20 g/1) such as beta-glucans. One example is scleroglucan, a polysaccharide produced by certain filamentous fungi (e.g. Sclerotinia.

Corticium, and Stromatina species) which, because of its physical characteristics, has been used as a lubricant and as a pressure-compensating material in oil drilling (Wang, Y., and B. Mc Neil. 1996. Scleroglucan. Critical Reviews in Biotechnology 16: 185-215).

Scleroglucan consists of a β( 1-3) linked glucose backbone with different degrees of β(l-6) glucose side groups. The presence of these side groups increases the solubility and prevents triple helix formation which, by consequence, decreases its ability to form gels. The viscosity of scleroglucan solutions shows high tolerance to pH (pH 1-11), temperature (constant between 10-90°C) and electrolyte change (e.g. 5% NaCl, 5% CaCl₂). Furthermore, its applications in the food industry for bodying, suspending, coating and gelling agents have been suggested and strong immune stimulatory, anti-tumour and anti-microbial activities have been reported (Kulicke, W.-M., A. I. Lettau, and H. Thielking. 1997, Correlation between immunological activity, molar mass, and molecular structure of different

(l→3)-β-D-glucans. Carbohydr. Res. 297: 135-143).

Remarkably, a class of filamentous fungi has now been identified and isolated which has been found to produce a fungal exopolysaccharide that exhibits characteristics that are attractive to the food industry. Two aspects of the EPS of interest are (a) its good texturing properties and (b) its ability to promote an immuno-stimulatory effect in in vitro and in vivo immunological assays. The fungal EPS could be incorporated into a health food (e.g. EPS as texturing fat replacer for low-calorie products or new immuno-stimulatory products) or provided alone for example as a food supplement.

Surprisingly, it has been found that these fungi are able to produce a remarkably high yield of a beta-glucan.

Accordingly, in a first aspect the present invention provides a method of producing a beta-glucan which comprises fermenting a suspension comprising a non-pathogenic saprophytic filamentous fungus and extracting a beta-glucan from the suspension.

In a second aspect the present invention provides use of a non-pathogenic saprophytic filamentous fungus or composition comprising it for providing a beta-glucan and thereby enhancing food structure, texture, stability or a combination thereof.

In a third aspect the invention provides use of a non-pathogenic saprophytic filamentous fungus or composition comprising it for providing a source of a beta-glucan and thereby providing nutrition.

In a forth aspect the invention provides use of a non-pathogenic saprophytic filamentous fungus or composition comprising it in the manufacture of a medicament or nutritional composition for the prevention or treatment of an immune disorder, tumour or microbial infection.

Preferably, an embodiment of a method of producing a beta-glucan comprises fermenting a non-pathogenic saprophytic filamentous fungus selected from the group which consists of Penicillium chermesinum, Penicillium ochrochloron, Rhizoctonia sp., Phoma sp., or a combination thereof. More preferably, at least three of these fungi are fermented together. More preferably all of these fungi are fermented together.

Preferably, an embodiment of a method of producing a beta-glucan comprises the step of fermenting for at least about 50 hours, more preferably about 80 hours to about 120 hours, even more preferably about 96 hours. Remarkably, it has now been found that if fermentation is carried out for this time, it provides the advantage that a high yield of beta-glucan is produced.

Preferably, an embodiment of a method of producing a beta-glucan comprises the step of fermenting a suspension in a medium comprising a component selected from the group which consists of NaN0₃, KH₂P0₄, MgS0₄, KC1 and yeast extract. More preferably it comprises at least three of these components. Most preferably it comprises all of these components. It has been found that a medium having these components provides the advantage that a high yield of beta-glucan is produced.

Preferably, an embodiment of a method of producing beta-glucans comprises the step of cultivating the fungus in minimal medium. Preferably, the medium comprises only glucose and salts and provides the advantage of enabling isolation of a highly pure polysaccharide at the expense of the production yield. This is because yeast extract contains polysaccharides that are difficult to separate from the EPS. Most preferably the medium comprises NaN0₃ (10 mM), KH₂P0₄ (1.5 g/1), MgS0₄ (0.5 g/1), KC1 (0.5), C₄H₁₂N₂0₆ (10 mM) glucose (60) adjusted to pH 4.7.

Preferably, an embodiment of use of a fungus according to an aspect of the invention comprises use of a fungus selected from the group which consists of Penicillium chermesinum, Penicillium ochrochloron, Rhizoctonia sp., Phoma sp., or a combination thereof.

Additional features and advantages of the present invention are described in, and will be apparent from the description of the presently preferred embodiments which are set out below.

In an embodiment, a method of producing a beta-glucan comprises fermenting a suspension which comprises a fungus in a medium of (g/1) NaN0₃ (3), KH₂P0₄ (1), MgS0₄ (0.5), KC1 (0.5), Yeast Extract (1.0), glucose (30) adjusted to pH 4.7. The fermentation is allowed to proceed for about 96 hours at about 28 °C with shaking at about 18rpm. In an alternative embodiment, strains which initially do not appear to produce polysaccharide are incubated for about 168 hours.

The following examples are given by way of illustration only and in no way should be construed as limiting the subject matter of the present application.

Example 1:

FUNGAL BETA-GLUCAN PRODUCTION:

The following fungal isolates were isolated and classified:

* * anamorph = asexual form, * teleomorph= sexual form N/A = not available.

Example 2

STANDARD POLYSACCHARIDE PRODUCTION

Media TB l (g/1) was used as follows: NaN0₃ (3), KH₂P0₄ (1), MgS0₄ (0.5), KC1 (0.5), Yeast Extract (1.0), glucose (30) adjusted to pH 4.7

Fermentation time was 96 h at 28°C with shaking at 180 rpm. For strains which initially did not seem to produce any polysaccharide the incubation was prolonged to 168 h.

Results of polysaccharide production were as follows:

Fungal strain Biomass Polysaccharide pH Specific

(g/i) (g/i) production

(g/g)

Slerottum ofucanicum NRRL 9.06 ± 2.06 1 1 .20 + 0.71 3.79 1 .24

3006

Botrttis ctnerea P3 2.64 ± 0.10 5.90 ± 0.57 4.35 2.23

Scler ottiiia sclerottorum P4 1 .16 + 0.16 1.61 ± 0.13 2.50 1.38

Fits ar turn culmorum P8 6.51 ± 1.05 0.82 + 0.13 7.70 0.13

Not identified P9 5.43 ± 0.53 1 .32 + 0.02 4.00 0.24

Penicillium chermesinum P28 4.08 ± 1 .17 0.68 + 0.1 1 3.30 0.17

Penicillium ochrochloron P45 10.53 ± 2.87 0.45 ± 0.07 3.50 0.04

Fusarmm sp. P58 8.60 ± 2.12 1 .25 ± 0.35 7.44 0.15 Sclerotinia sclerotiorum P62 2.10 ± 0.00 0.86 ± 0.00 3.80 0.41

Sclerotinia sclerotiorum P63 4.08 + 0.54 1.33 ± 0.04 3.30 0.33

Botritis fabae P65 19.70 ± 0.00 0.50 ± 0.00 4.94 0.03

Rhizoctonia fragariae P70 12.52 ± 0.40 1.55 ± 0.07 8.60 0.12

Colletotrichum acutatum P72 6.01 ± 0.89 1.05 ± 0.07 7.00 0.17

Pestalotia sp. P75 8.70 ± 0.28 1.90 ± 0.28 6.30 0.22

Colletotrichum sp. P80 12.00 + 1.95 0.65 ± 0.07 6.50 0.05

Colletotrichum sp. P81 5.10 ± 0.71 0.80 + 0.00 5.70 0.16

Rhizoctonia sp. P82 5.70 + 0.28 8.90 ± 1.56 6.50 1.56

Acre onium sp. P83 4.69 + 0.62 1.45 + 0.07 7.20 0.31

Acremonium sp. P84 5.50 ± 0.00 1.30 ± 0.00 7.20 0.24

Acremonium sp. P86 3.90 ± 0.71 l .OO ± 0.14 5.85 0.26

Acremonium sp. P90 8.08 ± 0.01 0.73 ± 0.18 4.40 0.09

Not identified P91 10.50 ± 0.14 1.28 + 0.31 6.83 0.12

Chaetomium sp. P94 8.30 ± 1.43 l .OO ± 0.28 7.40 0.12

Phoma herbarum P97 13.61 + 2.34 0.98 ± 0.22 7.50 0.07

Phoma sp. P98 1 1.01 ± 1.07 2.89 ± 0.01 8.00 0.26

P/70777-7 sp. P99 1 1.76 ± 1.66 0.66 ± 0.04 6.45 0.06

* Values are given at the time of maximum EPS production. Data are means of two independent experiments ±standard deviation.

Example 3

OPTIMIZED POLYSACCHARDE PRODUCTION

Polysaccharide production by Rhizoctonia sp. P82, Phoma sp. P98 and Penicillium chermesinum P28 were studied. The results were as follows:

A. Effect of carbon source cultivated on TB 1 : I. EPS production by Rhizoctonia sp. P82

Carbon source** Biomass Polysaccharide PH Specific

(g/i) (g/i) production

(g/g)

Glucose 3.74 + 0.80 18.55 + 0.57 5.48 4.96

Fructose 4.20 ± 0.58 21.10 + 0.89 5.60 5.02

Galactose 4.21 ± 0.19 16.67 ± 1.20 6.52 3.96 Xylose 3.45 ± 0.53 15.94 ± 2.42 6.07 4.63

Sorbitol 5.19 + 0.80 4.70 ± 0.21 6.16 0.91

Glycerol 5.25 ± 0.60 1.54 ± 0.42 6.15 0.29

Sucrose 4.03 + 0.59 14.07 ± 0.64 5.61 3.49

Maltose 4.07 + 0.32 12.22 ± 0.34 5.28 3.00

Lactose 4.63 ± 0.47 8.78 ± 0.59 6.34 1.90

Starch 5.77 ± 0.95 17.36 ± 0.69 6.26 3.01

* Values are given at the time of maximum EPS production. Data are means of three independent experiments ± standard deviation. ** Carbon sources were added to the medium at 30 g/1.

II. EPS production by Phoma sp. P98.

Carbon source** Biomass Polysaccharide PH Specific

(g/i) (g/i) production (g/g)

Glucose 1 1.99 ± 0.64 1.97 ± 1.22 7.31 0.16

Fructose 1 1.1 1 ± 0.76 1.22 ± 0.45 7.35 0.11

Galactose 10.35 ± 0.78 4.12 ± 0.03 7.44 0.40

Xylose 1 1.47 ± 1.40 2.57 ± 0.27 7.35 0.22

Sorbitol l i .π ± 0.69 7.54 ± 1.10 7.10 0.68

Glycerol 1 l .OO ± 0.37 0.63 ± 0.05 7.29 0.06

Sucrose 12.93 ± 0.44 2.91 ± 0.55 7.36 0.23

Maltose 12.50 ± 0.18 2.65 ± 0.98 6.92 0.21

Lactose 9.77 ± 0.01 1.06 ± 0.14 7.05 0.1 1

Starch 13.51 ± 1.65 2.28 ± 0.1 1 7.43 0.17

*Values are given at the time of maximum EPS production. Data are means of three independent experiments ± standard deviation. **Carbon sources were added to the medium at 30 g/1.

III. EPS production by Penicillium chermesinum P28*.

Carbon source** Biomass Polysaccharide PH Specific

(g/i) (g/i) production

(g/g)

Glucose 1 1.69 + 0.04 0.59 ± 0.13 3.51 0.05

Fructose 12.91 ± 1.20 0.46 ± 0.06 3.64 0.04

Galactose 8.64 ± 2.09 0.00 ± 0.00 5.23 0.00

SUBSTITUTE SHEET (RULE 26 Xylose 10.68 ± 0.06 0.41 ± 0.13 3.57 0.04

Sorbitol 8.58 ± 1.67 1.09 ± 0.01 5.07 0.13

Glycerol 13.06 ± 1.05 0.18 + 0.04 3.57 0.01

Sucrose 13.1 1 ± 0.80 0.59 ± 0.1 1 3.44 0.05

Maltose 10.90 ± 1.1 1 0.61 ± 0.16 3.53 0.06

Lactose 9.38 ± 0.34 0.00 ± 0.00 4.69 0.00

Starch 9.92 ± 2.04 0.50 ± 0.05 3.58 0.05

* Values are given at the time of maximum EPS production. Data are means of three independent experiments ± standard deviation. **Carbon sources were added to the medium at 30 g/1.

B. Effect of glucose concentration cultivated on TBl : I. EPS production by Rhizoctonia sp. P82*.

Glucose Biomass Polysaccharide pH Specific

(g/i) (g/i) (g/i) production

(g/g)

30 3.74 ± 0.80 18.55 ± 0.57 5.85 4.96

40 7.29 ± 0.42 21.40 ± 0.89 6.03 2.94

50 8.30 ± 0.74 30.20 ± 1.47 5.67 3.64

60 8.17 ± 1.34 35.26 ± 1.64 6.13 4.32

* Values are given at the time of maximum EPS production. Data are means of three independent experiments ± standard deviation.

II . EP S production by Phoma sp . P98 * .

Sorbitol Biomass Polysaccharide pH Specific

(g/i) (g/i) (g/i) production

(g/g)

30 8.60 ± 0.88 5.78 ± 0.61 7.22 0.67

40 12.08 ± 0.71 8.76 ± 0.40 7.12 0.73

50 13.22 ± 1.43 10.70 ± 0.48 7.13 0.81

60 16.47 ± 0.21 13.1 1 ± 0.33 7.56 0.80

Suprisingly, it can be seen from the results that increasing the concentration of the carbon source (glucose and sorbitol for Rhizoctonia sp. P82 and Phoma sp. P98. respectively), EPS production by both strains increased markedly (approx. 100% increase) reaching a maximum of 35.2 and 13.1 g/1, respectively.

C. Effect of nitrogen source cultivated on TBl : I. EPS production by Rhizoctonia sp. P82.

Nitrogen Biomass Polysaccharide PH Specific source (g/i) (g/i) production

(g/g)

NaN0₃ 3.74 ± 0.80 18.55 + 0.57 5.53 4.96

NH₄N0₃ 4.05 ± 0.29 13.07 ± 1.87 2.58 3.23

Urea 5.54 + 0.35 21.20 + 0.14 5.43 3.82

(NH₄)₂HP0₄ 3.09 ± 0.81 14.26 ± 0.52 2.44 4.61

(NH₄)₂S0₄ 2.39 ± 0.49 8.91 ± 0.58 2.23 3.73

II. EPS production by Phoma sp. P98.

Nitrogen Biomass Polysaccharide PH Specific source (g/i) (g/i) production

(g/g)

NaN0₃ 11.46 + 0.85 3.24 ± 0.63 7.22 0.28

NH₄N0₃ 6.12 + 0.33 1.17 ± 0.43 2.33 0.19

Urea 8.09 + 1.01 3.57 + 0.97 6.18 0.44

(NH₄)₂HP0₄ 6.53 + 0.44 0.00 ± 0.00 2.43 0.00 * Values are given at the time of maximum EPS production. Data are means of three independent experiments ± standard deviation.

Besides sodium nitrate, other nitrogen sources such as urea, ammonium nitrate, ammonium phosphate and ammonium sulphate were used. Remarkably, on urea, EPS production by Rhizoctonia sp. P82 and Phoma sp. P98 reached the same levels obtained on sodium nitrate.

Example 4

EPS P URIFICA TION AND CHARA CTERIZA TION The EPSs produced by Rhizoctonia sp. P82, Phoma sp. P98 and Penicillium chermesinum P28 were purified. The polysaccharides were exclusively constituted of sugars, thus indicating suprisingly high levels of purity. Both thin layer chromatography (TLC) and gas chromatography (GC) analysis showed that the EPSs from Rhizoctonia sp. P82 and Phoma sp. P98 were constituted of glucose only. In contrast, that from P. chermesinum P28 was constituted of galactose with traces of glucose.

The molecular weights (MW) of the EPSs from Rhizoctonia sp. and Phoma sp., estimated by gel permeation chromatography using a 100x1 cm Sepharose CL4B gel (Sigma) column, were both approximately 2- 10⁶ Da.

Determination of the position of the glucosidic linkages in the EPSs from Rhizoctonia sp. P82 and Phoma sp. P98 was carried out by GCms and GC after methylation, total hydrolysis, reduction and acetylation. The main products were identified by GCms analysis as glucitol 2,4-di-O-methyl- tetracetylated, glucitol 2,4,6-tri-O-methyl-triacetylated and glucitol 2,3,4,6- tetra-O-methyl-diacetylated indicating that both EPSs were characterised by monosaccharides linked with β-1,3 and β-1,6 linkages. In the case of the EPS from Phoma sp., the GC analyses showed three peaks in a quantitative ratio typical of a glucan with many branches; besides the above reaction products, the same type of analysis showed that the EPS from Rhizoctonia sp. gave rise to other reaction products such as penta- and esa-O-methyl-acetylated compounds which clearly indicated an uncompleted methylation.

Surprisingly, NMR analysis confirmed that both polysaccharides were pure, constituted of glucose only and characterised by β-1,3 and β-1,6 linkages.

Example 5

EPS IMMUNO-STIMULATORY EFFECTS

The EPSs from Rhizoctonia sp. P82 and Phoma sp. P98 were subjected to in vitro and in vivo experiments. A purified scleroglucan, obtained from S. glucanicum NRRL 3006, was used as a control. The purified EPSs were randomly broken in fragments of different molecular weights (from 1 - 10⁶ to MO⁴ Da) by sonication. The free glucose concentrations of the sonicated samples did not increase, thus indicating that no branches were broken. The experiments were carried out with EPSs at high MW (HMW, the native EPSs), medium MW (MMW, around 5- 10⁵ Da) and low MW (LMW, around 5- 10⁴ Da).

Immuno-stimulatory action was evaluated in vitro by determining effect on TNF-α production, phagocytosis induction, lymphocytes proliferation and IL- 2 production.

All the EPSs stimulated monocytes to produce TNF-α factor; its content increased with increased polysaccharide concentration and was maximum when medium and low MWs were used.

In order to assess the effect of the EPSs on phagocytosis, two methods

(Phagotest and Microfluoimetric Phagocytosis Assay) were used. The results gave a good indication that a high concentration of EPS improves phagocytosis.

In contrast, no significant effects were observed on lymphocyte proliferation and IL-2 production when the EPSs were added either alone or in combination with phytohemagglutinin (PHA). In addition, no cytotoxic effects were observed.

An in vivo study was carried out to assess immuno-stimulatory activity of the

EPS using MMW (around 5- 10⁵ Da) glucan from Rhizoctonia sp. P82.

Female mice were inoculated three times subcutaneously (SC) and/or orally (OR) with MMW EPS (2 mg/100 g weight) and Lactobacillus acidophilus (MO⁸ cells/100 g weight) after 1, 8 and 28 days. Bleedings were carried out after 13 and 33 days. In vivo immuno-stimulation was evaluated by comparing antibody production by an ELISA test.

All the mice that received OR bacteria (groups 3, 4 and 5) showed no increase in their antibody content, regardless of their glucan inoculation. However, differences in antibody production were observed among mice inoculated SC with bacteria. Furthermore, antibody levels of mice that received SC only bacteria were significantly higher (P<0.01, by Tukey Test) than those that had received glucan and bacteria both SC and glucan OR and bacteria SC.

Interestingly, the results indicate that the EPS from Rhizoctonia sp. Gives rise to a decrease in antibody concentration. Remarkably, it can be concluded from this that the glucan from Rhizoctonia sp. causes activation of an antimicrobial activity of monocytes (see the effects described above relating to TNF-α production and phagocytosis induction) with a consequent reduction in the bacterial number leading, in turn, to a consistent reduction in antibody production.

In conclusion, the three filamentous fungi Rhizoctonia sp P82, Phoma sp. P98 and Penicillium chermesinum P28 have a suprisingly good ability to produce extracellular polysaccharides of potential interest. In particular, Rhizoctonia sp. P82 is interesting in view of its short time required for fermentation, its high level of EPS production and its absence of β-glucanase activity during the EPS production phase. Furthermore, its EPS, as well as that from Phoma sp. P98, is a glucan characterised by β-1,3 and β-1,6 linkages. In addition, results relating to immuno-stimulatory effects of the glucan produced by Rhizoctonia sp. P82 indicate the possibility of a good stimulatory activity.

It should be understood that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present invention and without diminishing its attendant advantages. It is therefore intended that such changes and modifications be covered by the appended claims.

Claims

1. A method of producing a beta-glucan which comprises fermenting a suspension comprising a non-pathogenic saprophytic filamentous fungus and extracting a beta-glucan from the suspension.

2. A method according to claim 1 wherein the non-pathogenic saprophytic filamentous fungus is selected from the group which consists of Penicillium chermesinum, Penicillium ochrochloron, Rhizoctonia sp., Phoma sp., or a combination thereof.

3. A method according to claim 1 or 2 wherein the non-pathogenic saprophytic filamentous fungi Penicillium chermesinum. Penicillium ochrochloron, Rhizoctonia sp. and Phoma sp. are fermented together.

4. A method according to any preceding claim wherein the step of fermentation is carried out for at least about 50 hours.

5. A method according to any preceding claim wherein the step of fermenting is carried out in a medium comprising a component selected from the group which consists of NaN0₃, KH₂P0₄, MgS0₄, KC1 and yeast extract.

6. A method according to any preceding claim wherein the step of fermenting is carried out by cultivating the fungus in minimal medium which comprises only glucose and salts.

7. A method according to any preceding claim wherein the step of fermenting is carried out by cultivating the fungus in a medium which comprises NaN0₃ (10 mM), KH₂P0₄ (1.5 g/1), MgS0₄ (0.5 g/1), KC1 (0.5), C₄H₁₂N₂0₆ (10 mM) glucose (60) adjusted to pH 4.7.

8. Use of a non-pathogenic saprophytic filamentous fungus or composition comprising it for providing a beta-glucan and thereby enhancing food structure, texture, stability or a combination thereof.