Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing a carotene nucleus
Reexamination Certificate
2001-12-20
2004-11-02
Prats, Francisco (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing a carotene nucleus
C435S171000, C585S351000, C585S803000, C585S836000
Reexamination Certificate
active
06812001
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the field of microbially produced carotenoid compounds.
BACKGROUND OF THE INVENTION
Currently, &bgr;-carotene crystals with a high purity (96% or higher) are produced by chemical synthesis. When derived from a natural source, &bgr;-carotene mostly is in the form of an oily extract (palm oil, algal oil). Although it is also possible to obtain &bgr;-carotene crystals from natural sources, such as vegetables (for example carrots) or micro-organisms (for example algae (Dunaliella) or fungi (Blakeslea), the currently available processes to obtain relatively pure crystals from said natural sources have important disadvantages.
Purification of &bgr;-carotene crystals from natural sources comprises extraction of the &bgr;-carotene from said source with a suitable extractant, optionally followed by additional purification steps until the desired purity is obtained.
The extraction is carried out with various extractants: organic solvents, such as ethyl acetate, butyl acetate, hexane; vegetable oils, or supercritical fluids, such as propane, ethylene, CO
2
. Subsequently, the &bgr;-carotene crystals can be directly crystallized from the extract obtained after solvent extraction of said natural source, e.g. by evaporation of the solvent.
A main disadvantage of a solvent extraction process is that the &bgr;-carotene crystals should firstly be solubilized in a solvent, whereupon, after separating the biomass residue from the &bgr;-carotene-containing solvent, the &bgr;-carotene should be crystallized again. In addition, a considerable loss of &bgr;-carotene can easily occur.
To circumvent the use of the large amounts of solvent which are necessary to solubilize the &bgr;-carotene, it would be desirable to directly isolate &bgr;-carotene, or any other carotenoid, in a crystalline form from microbial biomass.
DESCRIPTION OF THE INVENTION
The present invention describes a process for the isolation of a carotenoid compound from microbial biomass. The process of the invention is applicable to microbial biomass in which carotenoid compounds are present in a crystalline form. According to the process of the present invention, the carotenoid crystals are directly separated from the microbial biomass. An important advantage of the process of the invention is that it is applicable without using large amounts of solvent. In particular, the amount of solvent used in the process of the invention is substantially reduced as compared to the large amounts of solvent which are needed to solubilize the carotenoid using a conventional solvent-extraction process.
The process of the invention essentially comprises the steps of disrupting the microbial cell walls, separating cellular debris from the carotenoid-containing residue, including a wash of either the microbial biomass, the disrupted cell mass or the carotenoid-containing residue with a solvent suitable to remove lipid, floating the obtained carotenoid crystals in water, and optionally, further purifying the crystals.
The following steps of the process of the invention are described in more detail.
The carotenoid-containing microorganism can be a bacterial, a yeast, a fungal or an algal microorganism. Preferably, the carotenoid-containing microorganism is a yeast, a fungus or an algae. More preferably, it is a yeast of the genus
Phaffia
, a fungus of the order
Mucorales
or an algae of the genus
Dunaliella.
Microbial carotenoid-containing biomass is obtained from any suitable fermentation of a carotenoid-producing microorganism of choice, as specified above.
The microbial biomass which is subjected to the process of the invention may be in the form of a wet cell cake or in a dry form. For economical reasons, the use of a wet cell cake is preferred. Dry biomass may for instance be in the form of an extrudate, as is described in WO 97/36996.
Cell disruption may occur via methods known in the art. The disruption may occur physically (mechanically), enzymatically and/or chemically. Preferably, cell disruption is performed by mechanical means. Mechanical disruption may occur for instance by homogenizing microbial biomass in a homogenizer under high pressure or using a bead mill or by ultrasonification. Chemical disruption may occur in a low or high pH environment, or by addition of a solvent such as octanol. Enzymatical disruption may occur by action of an enzyme or enzyme mixture degrading constituents of the microbial cell wall.
For efficient cell disruption, the biomass typically may have a dry matter content of about 10 to about 200 g/l. Conveniently, the fermentation broth directly obtained after fermentation is used, having a dry matter content of about 50 g/l. When the starting material is dry biomass, said biomass is mixed with a sufficient amount of water to obtain a dry matter content of about 10 to about 200 g/l, as specified above.
To improve the yield of the recovery process of the invention, an organic solvent not miscible with water may optionally be added to the disrupted cell mass before any further processing steps are taken. Depending on the type of disruption process being applied, addition of said solvent comprises an addition occurring before, during or after cell disruption. For instance, in the case that the cells are disrupted by homogenizing, the solvent preferably is added after the disruption step. The oil or the solvent is added in an amount of 1% to 100% of the amount of biomass suspension or disrupted cell mass, preferably 3% to 10% of the amount of biomass suspension or disrupted cell mass. A suitable organic solvent not miscible with water is, for instance, an oil, hexane or ethyl acetate. Preferably, an oil is added to the disrupted cell mass. Examples of suitable oils are vegetable oils such as soybean oil.
From the resulting disrupted cell mass, a substantial part of the cellular debris is removed by decantation or centrifugation. Preferably, a centrifugation step is applied. Said centrifugation results in a solid upper, a liquid middle and a solid under layer, the solid upper layer containing the carotenoid crystals, also called the carotenoid-containing residue. Only a very small amount of carotenoid is lost in this step.
Optionally, the solid upper layer with the carotenoid-containing residue, said residue essentially consisting of carotenoid crystals, microbial lipid and remaining cellular debris, is washed with water one or more times, to remove additional cellular debris. Said water optionally may contain a salt, such as sodium chloride. The salt may be present in a concentration up to 25% (w/w).
The process of the invention further includes a washing step with a suitable solvent to remove a substantial part of the microbial lipid and, optionally, any oil previously added to the disrupted cells.
A suitable solvent for lipid removal is a lipid- and water-miscible solvent in which the crystalline carotenoid has a low solubility. Preferably, said solvent is a lower alcohol, such as methanol, ethanol, isopropanol, or acetone. More preferably, said solvent is ethanol. It should be noted that the amount of solvent necessary for lipid removal is substantially lower than the amount of solvent necessary for solvent-extraction of a carotenoid from microbial biomass.
In a preferred embodiment of the invention, the carotenoid-containing residue obtained after separation of the cellular debris is washed with said suitable solvent for lipid removal. Said washing is carried out by stirring the carotenoid-containing residue for a convenient time period, e.g for about 10 minutes, with said solvent and recovering the solid under layer after centrifugation. Optionally, said washing with said solvent is repeated one or more times.
In another embodiment of the invention, lipid can be removed from the microbial biomass before cell disruption takes place. This alternative embodiment especially is applicable if the starting biomass material is in a dry form. Typically, dry biomass is suspended in the solvent of choice in an amount of 10 to 400 g biomass per liter solvent. To incre
De Pater Robertus Mattheus
Sibeijn Mieke
DSM N.V.
Morrison & Foerster / LLP
Prats Francisco
LandOfFree
Isolation of carotenoid crystals from microbial biomass does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Isolation of carotenoid crystals from microbial biomass, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Isolation of carotenoid crystals from microbial biomass will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3292953