Abstract:
A method for producing a beta-glucan from a non-pathogenic saprophytic filamentous fuingus or composition that contains it. Also, methods for providing this beta-glucan in a food product to improve structure, texture, stability or combinations thereof, in a food product to provide nutrition or in the manufacture of a medicament or nutritional composition for the prevention or treatment of an immune disorder, tumor or microbial infection.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
       [0001]     This application is a continuation of U.S. application Ser. No. 10/395,191 filed Mar. 25, 2003, which is a continuation of U.S. application Ser. No. 10/236,991 filed Sep. 5, 2002, which is a continuation of the U.S. National Stage of International Application No. PCT/EPO 1/03100 filed Mar. 20, 2001, the entire contents of all of which are expressly incorporated herein by reference thereto. 
     
    
     TECHNICAL FIELD  
       [0002]     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, tumor or microbial infection.  
       BACKGROUND ART  
       [0003]     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, the characterization of exopolysaccharide (“EPS”) production at a biochemical as well as at a genetic level has been studied. An advantage of EPS is that it can be secreted by food micro-organisms during fermentation, but using EPS produced by micro-organisms gives rise to the problem that the level of production is very low (50-500 mg/l) and that once the EPS is extracted it loses its texturing properties.  
         [0004]     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 processors in the food-industry: viscosifing, emulsifying, stabilising, cryoprotectant and immune-stimulating activities.  
         [0005]     Remarkably, it has been found that fungi can produce high amounts of biopolymers (20 g/l) 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).  
         [0006]     Scleroglucan consists of a β(1-3) linked glucose backbone with different degrees of β(1-6) glucose side groups. The presence of these side groups increases the solubility and prevents triple helix formation that, 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% CaCl 2 ). Furthermore, its applications in the food industry for bodying, suspending, coating and gelling agents have been suggested and strong immune stimulatory, anti-tumor 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 (1→3)-β-D-glucans. Carbohydr. Res. 297: 135-143).  
         [0007]     As there is a need for these type materials in the food industry, they have been further investigated by the present inventors, and this invention now has identified unexpected benefits in food processing operations due to the use of these materials.  
       SUMMARY IF THE INVENTION  
       [0008]     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.  
         [0009]     Surprisingly, it has also been found that these fungi are able to produce a remarkably high yield of a beta-glucan.  
         [0010]     Accordingly, in a first aspect, the present invention provides a method for producing a beta-glucan which comprises: fermenting a suspension comprising a non-pathogenic saprophytic filamentous fungus selected from the group consisting of  Penicillium chermesinum, Penicillium ochrochloron, Rhizoctonia  sp.,  Phoma  sp., or a combination thereof in a minimal medium consisting essentially of glucose and salts; and extracting the beta-glucan from the fermented suspension.  
         [0011]     Preferably, the fermentation is carried out for at least about 50 hours. The fermentation medium may additionally comprise a component selected from the group consisting of NaNO 3 , KH 2 PO 4 , MgSO 4 , KCl, and yeast extract, such that NaNO 3  (10 mM), KH 2 PO 4  (1.5 g/l), MgSO 4  (0.5 g/l), KCl (0.5 g/l), C 4 H 12 N 2 O 6  (10 mM), and glucose (60 g/l) are present in the fermentation medium. The pH of the medium may preferably be adjusted to a pH of 4.7.  
         [0012]     According to one preferred embodiment, the fungi Penicillium chermesinum, Penicillium ochrochloron, Rhizoctonia sp. and Phoma sp. may be fermented together. The fermentation may be carried out for at least about 50 hours, and the medium may additionally comprise a component selected from the group consisting of NaNO 3 , KH 2 PO 4 , MgSO 4 , KCl, and yeast extract.  
         [0013]     The present invention also provides for enhancing the structure, texture, or stability of a food product by adding an effective amount of the beta-glucan produced according to the present method to the food product. Similarly, the beta-glucan produced by the present method may be added to a nutritional composition to provide enhanced nutrition, or to a medicament for prevention or treatment of an immune disorder, tumor, or microbial infection.  
     
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS  
       [0014]     One or more of a non-pathogenic saprophytic filamentous fungus selected from the group consisting of  Penicillium chermesinum, Penicillium ochrochloron, Rhizoctonia  sp.,  Phoma  sp., and combinations thereof is fermented to form the beta-glucan. Preferably, at least three of these fungi are fermented together. More preferably all of these fungi are fermented together.  
         [0015]     The fermenting step is conducted for at least about 50 hours, preferably for about 80 hours to about 120 hours, and even more preferably for about 96 hours. These times are advantageous for obtaining high yields of beta-glucan.  
         [0016]     The fermenting step is advantageously conducted in suspension in a medium comprising at least one component selected from the group consisting of NaNO 3 , KH 2 PO 4 , MgSO 4 , KCl and yeast extract. Preferably, at least two or three of these components are used and most preferably all these components are used together to provide the best yields of beta-glucan. Advantageously, the beta-glucan is added to a food product, a nutritional composition, or a medicament.  
         [0017]     Preferably, the fungus is cultivated in a minimal medium. More preferably, the medium consists essentially of 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 NaNO 3  (10 mM), KH 2 PO 4  (1.5 g/l), MgSO 4  (0.5 g/1), KCl (0.5), C 4 H 12 N 2 O 6  (10 mM) glucose (60) and has a pH of 4.7.  
         [0018]     The suitable fungus that can be used according to the invention includes those selected from the group consisting of  Penicillium chermesinum, Penicillium ochrochloron, Rhizoctonia  sp.,  Phoma  sp., or a combination thereof.  
         [0019]     Additional features and advantages of the present invention are described in, and will be apparent from the description of the most preferred embodiments which are set out below and in the examples.  
         [0020]     In one preferred embodiment, beta-glucans are produced by fermenting a suspension which comprises a fungus in a medium of (g/l) NaNO 3  (3), KH 2 PO 4  (1), MgSO 4  (0.5), KCl (0.5), Yeast Extract (1.0), and glucose (30) with the pH of medium adjusted to 4.7. The fermentation is allowed to proceed for about 96 hours at about 28° C. with shaking at about 18 rpm. In an alternative embodiment, strains which initially do not appear to produce the polysaccharide are incubated for about 168 hours and then are added to the medium under the previously described conditions.  
       EXAMPLES  
       [0021]     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  
       [0022]     The following fungal isolates were isolated and classified:  
                                       Lab-isolate   “Italian”, public name   CBS identification                   P28     Penicillium chermesinum       Penicillium glabrum                 (teleomorph*)       P45     Penicillium ochrochloron       Eupenicillium euglaucum                 (anamorph**)       P82     Rhizoctonia  sp.     Botryosphaeria rhodina                 (teleomorph)/                 Lasiodiplodia theobromae                 (anamorph)       P98     Phoma  sp.   N/A       VT13     Phoma  sp.   N/A       VT14     Phoma  sp.   N/A                 **anamorph = asexual form,            *teleomorph = sexual form            N/A = not available.             
 
       Example 2  
     Standard Polysaccharide Production  
       [0023]     Media TB1 (g/l) was used as follows: NaNO 3  (3), KH 2 PO 4  (1), MgSO 4  (0.5), KCl (0.5), Yeast Extract (1.0), and glucose (30) with the pH adjusted to 4.7.  
         [0024]     The 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.  
         [0025]     Results of polysaccharide production were as follows:  
                                                                                     Specific           Biomass   Polysaccharide       production       Fungal strain   (g/l)   (g/l)   pH   (g/g)                                  Slerotium glucanicum  NRRL 3006   9.06 ± 2.06   11.20 ± 0.71    3.79   1.24         Botritis cinerea  P3   2.64 ± 0.10   5.90 ± 0.57   4.35   2.23         Sclerotinia sclerotiorum  P4   1.16 ± 0.16   1.61 ± 0.13   2.50   1.38         Fusarium 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 ± 0.11   3.30   0.17         Penicillium ochrochloron  P45   10.53 ± 2.87    0.45 ± 0.07   3.50   0.04         Fusarium  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         Acremonium  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   1.00 ± 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   1.00 ± 0.28   7.40   0.12         Phoma herbarum  P97   13.61 ± 2.34    0.98 ± 0.22   7.50   0.07         Phoma  sp. P98   11.01 ± 1.07    2.89 ± 0.01   8.00   0.26         Phoma  sp. P99   11.76 ± 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  
       [0026]     Polysaccharide production by  Rhizoctonia  sp. P82,  Phoma  sp. P98 and  Penicillium chermesinum  P28 were studied. The results were as follows:  
         [0027]     A. Effect of Carbon Source Cultivated on TB 1:  
                                                                                                                                                   Specific           Biomass   Polysaccharide       production       Carbon source**   (g/l)   (g/l)   pH   (g/g)                                I. EPS production by  Rhizoctonia  sp. P82            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            II. EPS production by  Phoma  sp. P98.            Glucose   11.99 ± 0.64   1.97 ± 1.22   7.31   0.16       Fructose   11.11 ± 0.76   1.22 ± 0.45   7.35   0.11       Galactose   10.35 ± 0.78   4.12 ± 0.03   7.44   0.40       Xylose   11.47 ± 1.40   2.57 ± 0.27   7.35   0.22       Sorbitol   11.17 ± 0.69   7.54 ± 1.10   7.10   0.68       Glycerol   11.00 ± 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   0.11       Starch   13.51 ± 1.65   2.28 ± 0.11   7.43   0.17            III. EPS production by  Penicillium chermesinum  P28*.            Glucose   11.69 ± 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       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   13.11 ± 0.80   0.59 ± 0.11   3.44   0.05       Maltose   10.90 ± 1.11   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/l.             
 
         [0028]     B. Effect of Glucose Concentration Cultivated on TB1:  
                                                                                                                   Biomass   Polysaccharide       Specific production           (g/l)   (g/l)   pH   (g/g)                                    I. EPS production by  Rhizoctonia  sp. P82*.            Glucose (g/l)                       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            II. EPS production by  Phoma  sp. P98*.            Sorbitol (g/l)                       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   13.11 ± 0.33   7.56   0.80                 *Values are given at the time of maximum EPS production. Data are means of three independent experiments ± standard deviation.             
 
         [0029]     Surprisingly, 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/l, respectively.  
         [0030]     C. Effect of Nitrogen Source Cultivated on TB1:  
                                                                                                                       Specific       Nitrogen   Biomass   Polysaccharide       production       source   (g/l)   (g/l)   PH   (g/g)                                I. EPS production by  Rhizoctonia  sp. P82.*            NaNO 3     3.74 ± 0.80   18.55 ± 0.57    5.53   4.96       NH 4 NO 3     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       (NH 4 ) 2 HPO 4     3.09 ± 0.81   14.26 ± 0.52    2.44   4.61       (NH 4 ) 2 SO 4     2.39 ± 0.49   8.91 ± 0.58   2.23   3.73            II. EPS production by  Phoma  sp. P98*            NaNO 3     11.46 ± 0.85    3.24 ± 0.63   7.22   0.28       NH 4 NO 3     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       (NH 4 ) 2 HPO 4     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.             
 
         [0031]     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 Purification and Characterization  
       [0032]     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.  
         [0033]     The molecular weights (MW) of the EPSs from  Rhizoctonia  sp. and  Phoma  sp., estimated by gel permeation chromatography using a 100×1 cm Sepharose CL4B gel (Sigma) column, were both approximately 2·10 6  Da.  
         [0034]     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.  
         [0035]     Surprisingly, NMR analysis confirmed that both polysaccharides were pure, constituted of glucose only and characterized by β-1,3 and β-1,6 linkages.  
       Example 5  
     EPS Immuno-Stimulatory Effects  
       [0036]     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 1·10 6  to 1·10 4  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 5·10 5  Da) and low MW (LMW, around 5·10 4  Da).  
         [0037]     Immuno-stimulatory action was evaluated in vitro by determining effect on TNF-α production, phagocytosis induction, lymphocytes proliferation and IL-2 production.  
         [0038]     All the EPSs stimulated monocytes to produce TNF-a factor; its content increased with increased polysaccharide concentration and was maximum when medium and low MWs were used.  
         [0039]     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.  
         [0040]     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.  
         [0041]     An in vivo study was carried out to assess immuno-stimulatory activity of the EPS using MMW (around 5·10 5  Da) glucan from  Rhizoctonia  sp. P82.  
         [0042]     Female mice were inoculated three times subcutaneously (SC) and/or orally (OR) with MMW EPS (2 mg/100 g weight) and  Lactobacillus acidophilus  (1·10 8  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.  
         [0043]     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&lt;0.01, by Tukey Test) than those that had received glucan and bacteria both SC and glucan OR and bacteria SC.  
         [0044]     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.  
         [0045]     In conclusion, the three filamentous fungi  Rhizoctonia  sp. P82,  Phoma  sp. P98 and  Penicillium chermesinum  P28 have a surprisingly 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.  
         [0046]     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.