Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1998-06-24
2002-09-03
Prats, Francisco (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S099000, C435S095000, C435S072000, C435S169000, C435S170000, C435S171000, C435S259000, C426S060000, C426S062000, C426S656000, C424S234100, C424S274100, C424S278100, C424S282100, C424S093500, C424S093510, C514S054000, C514S777000
Reexamination Certificate
active
06444448
ABSTRACT:
This invention relates to biologically active &bgr;-glucan-mannan preparations and to methods for their isolation. In particular, the invention relates to &bgr;-glucan-mannan preparations including &bgr; (1-3) glucan, produced from microorganisms including but not limited to yeasts, and to methods for producing the &bgr;-glucan-mannan preparations which avoid the use of concentrated alkali or acid.
BACKGROUND OF THE INVENTION
Polysaccharides are widely distributed in nature, and are particularly important for their role in maintaining the structural integrity of bacterial fungal, and plant cells. Glucans are polymers of D-glucose, and the D-glucose units may be linked together in a variety of ways. For example, glucans with 1-3, 1-4, 1-6, and 1-2 linkages are all known. The variety of linkages possible means that glucans are normally highly branched compounds. Because of their chemical properties, glucans have found a wide variety of uses in the chemical, food and pharmaceutical industries. For example, they are useful as viscosity imparting agents, emulsifiers, fibers, films, coating substances, supports for affinity chromatography and gel electrophoresis, in cell culture media, as filter pads, and in cement. They are also widely used as food thickeners and as a source of dietary fibre, and as carriers and coating agents in pharmaceutical products.
Glucans, particularly &bgr; (1-3)-glucans, have been very extensively studied, and in addition to the foregoing, have been shown to have a variety of pharmacological activities, including but not limited to anti-cholesterolaemic activity, hypoglycaemic activity, acceleration of heavy metal excretion, and stimulation of the immune system. The immunostimulatory activity of &bgr;-glucans has led to suggestions that they are useful as anti-cancer agents or in the treatment of HIV infection, as agents for stimulation of wound healing, or as anti-infective agents for use either alone or in conjunction with antibiotics.
The &bgr;-glucans can also induce the resistance response to disease in plants, such as phytoalexin production and wilting. This has led to suggestions that they can be used as anti-infective agents and growth promoters in plants.
Because of the problems entailed in intensive poultry, animal, fish or crustacean production, the use of &bgr;-glucans as an additive to these feeds, to reduce incidence of infection and consequently to promote growth and to reduce the need for antibiotics, has also been proposed.
&bgr; (1-3)-glucan is an important cell wall component of yeast cells. The cell wall of
Saccharaomyces cerevisiae
is primarily composed of &bgr;-linked glucan, which is mainly a backbone of &bgr; (1-3)-linked glucose units, with a minor component of inter and intra molecular branching via &bgr; (1-6)-linkages. Because of the very wide use of yeasts in the food and brewing industry, as well as in the production of industrial-grade alcohol, spent yeast cells are a major industrial by-product. Yeast-derived products themselves have considerable. commercial value, for example in such products as yeast extracts, flavouring agents, flavour potentiators such as guanosine monophosphate and inosine monophosphate, and in the manufacture of enzymes, fine chemicals and products for use in the biochemical and pharmaceutical industries, such as trehalose, thymidine, nucleosides and nucleotides, etc. Waste yeast from the brewing industry is a major source of &bgr;-glucans.
In addition, other species of yeast are also useful as a source of &bgr;-glucans, including but not limited to other yeast strains of
Saccharomyces cerevisiae, Kluyveromyces fragilis
, and Candida strains such as
Candida utilis
. All of these yeast strains can be produced using culture in food grade nutrients either by batch fermentation or continuous fermentation. Many other species of microorganisms, including bacteria, fungi and unicellular algae, have been reported in the art as a source of &bgr;-glucans.
The purification of &bgr;-glucans from yeast and other organisms has been extensively investigated, and a variety of methods is known. Most of these rely on the insolubility of &bgr; (1-3)-glucan in alkali or in organic solvents. The principal known methods are:
(a) High temperature extraction with concentrated sodium hydroxide, followed by high temperature extraction with acid and precipitation with ethanol (see for example Manners, D. J. et al., Biochem. J. 135 19-30 (1973), Jamas, S. et al., U.S. Pat. Nos. 4,810,646, No. 5,028,703, and No. 5,250,436 and Australian Patent No. 628752. by Phillips Petroleum Company). Many of these protocols require preliminary homogenisation of the yeast cells, and many require multiple repetition of each extraction step.
(b) Extraction with concentrated sodium hydroxide, followed by high temperature acid extraction and enzyme treatment to modify or purify the glucan (see for example Czech Patent Application No. 890038 by Masler, L. et al. which reports purification of &bgr;-D-glucan by alkali-acid extraction, followed by treatment with enzymes having amylase activity).
(c) Extraction of yeast cell wall preparations resulting from autolysis or enzyme degradation of yeast with concentrated phenol: water (1:1) (see for example U.S. Pat. No. 4,138,479 by Truscheit, E. et al.).
(d) Extraction with organic solvents such as isopropanol, ethanol, acetone, or methanol either alone or in the presence of alkali (see for example Japanese Patent publications No. 7051081, 6340701, 5295003, and 3002202; European Patent Application No. 515216).
Acid treatment is known to reduce the number of &bgr; (1-6)-linkages in the glucan material and this results in an increase in viscosity.
The cell wall of yeast is mainly composed of:
(i) fibrillar, alkali insoluble &bgr; (1-3)-linked glucan, with side branches of &bgr; (1-6)-linked glucan.
(ii) alkali-soluble &bgr; (1-3)-linked glucan with side branches of &bgr; (1-6)-linked glucan.
(iii) amorphous acid-soluble &bgr; (1-6)-glucan, with intermittent &bgr; (1-3)-linkages.
(iv) amorphous alkali-soluble mannan linked to proteins.
The fibrillar glucan component is located adjacent to the yeast plasma membrane, and is covered externally by an amorphous layer composed mainly of mannoproteins (Kopecka M. et al., J. Cell Biol., 62 68-76 (1974)). Particulate material (zymosan) isolated from the cell walls of
Saccharomyces cerevisiae
is known to have the ability to act as a non-specific immune stimulant. The biological activity of yeast cell wall particulates is largely attributed to the presence of &bgr; (1-3)-linked glucan, but the other two forms of glucan and mannan also have some ability to stimulate the immune system. Mannan is reported to mediate the adsorption and phagocytosis of particulate material, such as insoluble &bgr; (1-3)-glucan, by cells of the immune system (Giaimis, J., et al., Journal Of Leukocyte Biology 54 564-571 (1993); Sun-Sang, J., et al., Journal Of Cell Biology 96 160-166 (1983)).
Existing methods to isolate the &bgr;-glucans commonly use a multi-step alkali-acid extraction process (Manners, D. J., et al., J. Gen. Micro. 80 411-417 (1974)). The alkali extraction steps remove most of the amorphous mannoprotein and glucan material, and the subsequent acid extraction steps remove the glycogen and most of the &bgr; (1-6)-side branches from the fibrillar predominantly &bgr; (1-3) linked glucan. A final solvent extraction step is sometimes used to remove lipids.
It is clear that, given the retail price of glucan for some applications, the cost of producing glucan using existing published or patented methods is not commercially viable. These methods have the following problems:
(i) They are aimed at only producing the fibrillar, alkali-insoluble form of glucan. Other forms of glucan and the mannan present in the cell wall are removed as by-products of the process. These represent an additional amount of glucan, which could have significantly increased the glucan yield, and which may be functionally important.
(ii) The existing processes require significant capital investments.
(iii)
Gilbert Robert White
Kulandai Joseph
Langeris Willem Hendrik
Sime Keith James
Smith Craig Gordon
Carlton and United Breweries Limited
Prats Francisco
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