The present invention is directed to methods and apparatus for, and products from disrupting, spray drying, extracting and hydrolyzing Saccharomyces cerevisiae (yeast) to produce Beta (β)-glucans, chitin and mannans (mannoproteins). The process and apparatus feature critical, supercritical, or near critical fluids with or without cosolvents for disruption of yeast, removal of intracellular proteins, enzymes and nucleic acids, extraction of lipids and making yeast cell wall nanoparticles.
This application discloses a number of improvements and enhancements to supercritical fluid disruption and extraction from microbial cells disclosed in U.S. Pat. No. 5,380,826 by Castor et al. (1995), which is hereby incorporated by reference in its entirety.
This application discloses a number of improvements and enhancements to method for size reduction of proteins and apparatus disclosed in U.S. Pat. No. 6,051,694, Castor et al. (2000), which is hereby incorporated by reference in its entirety.
Yeast cell walls consist of 70% neutral carbohydrate (polysaccharides), 7% amino sugars, 15% lipids and 0.8% phosphorous (Vega et al., 1986). The three main polysaccharide groups are β-glucans, polymers of mannose (mannoproteins known as mannans), around 60%. 40% and 2% respectively.
These polysaccharides are finding wide benefits in food, pet-food and feed products as well as dietary supplements. Insoluble β-glucans are reported to have immune modulation effects against infectious disease and cancer and enhanced antibiotic efficiency on infections with antibiotic resistant bacteria. β-glucans from Bakers' yeast have received GRAS status from the FDA in 1997 and are regulated in Europe as a “novel food.” It has also been shown that mannan improves gastrointestinal health by preventing of pathogens to host's cells.
In this invention, SuperFluids carbon dioxide can be used to disrupt Saccharomyces cerevisiae (Yeast) per “Supercritical Fluid Disruption and Extraction from Microbial Cells.” U.S. Pat. No. 5,380,826 by Castor et al. (1995). SuperFluids are supercritical fluids, critical fluids and/or near-critical fluids with or without polar cosolvents. This '826 patent is incorporated in full by reference in this disclosure.
Yeast in a slurry is first saturated with SuperFluids CO2 at operating pressures between 2,000 and 5,000 psig and temperatures between 20 and 60° C. After saturation, the yeast slurry is rapidly decompressed into a decompression chamber. As a result of expansive forces, yeast is disrupted and intracellular proteins, enzymes and nucleic acids are released and can be recovered.
In an embodiment of this invention, during decompression, the yeast solution can be heated so the liquid solvent (water) evaporates, and the disrupted yeast is dried into a powder as in a spray drier. In another embodiment, yeast can also be decompressed into a fully or partially evacuated chamber to achieve a spray drying effect. A combination of heat and low pressure can be utilized to produce a spray-dried disrupted yeast powder.
In another embodiment of this invention, SuperFluids CO2 at appropriate conditions of temperature and pressure can then be used to extract and remove lipids from the spay-dried yeast powder. Spray dried disrupted yeast powder is contacted with SuperFluids CO2 at operating pressures between 2,000 and 20,000 psig and temperatures between 20 and 100° C. to solubilize and remove lipids.
In another embodiment of this invention, the lipid-reduced, disrupted, spray-dried yeast powder saturated with SuperFluids C02 is rapidly expanded to produce yeast wall nanoparticles. This process is similar to “Method for Size Reduction of Proteins,” U.S. Pat. No. 6,051,694, Castor et al. (2000). This '694 patent is incorporated in full by reference in this disclosure.
In another embodiment of this invention, the lipid-reduced, disrupted, spray-dried yeast nanoparticles are processed by enzymatic cleavage to produce fractions of β-glucans, chitins and mannans.
In another embodiment of this invention, the lipid-reduced, disrupted, spray-dried yeast nanoparticles are processed by hydrolysis to produce fractions of β-glucans, chitins and mannans.
In another embodiment of this invention, the lipid-reduced, disrupted, spray-dried yeast nanoparticles are processed by a combination of acid hydrolysis and enzymatic cleavage to produce fractions of β-glucans, chitins and mannans.
These and other features of the invention are described in greater detail below.
Yeast in the form of slurry 210 is introduced to a SuperFluids (SFS) chamber at specified temperature and pressure sufficient for the SFS 220 to penetrate the cell walls of the yeast and saturate the yeast with SFS in 230. SFS used includes carbon dioxide, nitrous oxide, propane, alkanes and fluorocarbons. A preferred SFS is carbon dioxide. Pressures range from 500 psig to 5,000 psig. A preferred pressure is 3,000 psig. Temperatures range from 10° C. to 60° C. A preferred temperature is 40° C.
The SFS saturated yeast is rapidly decompressed via a back-pressure regulator 240 through a high-pressure single fluid nozzle (500 psig to 5,000 psig) into chamber 250 which also acts as spray drier that is exhausted by vent 260 which can be connected to a vacuum source. As a result of decompression, yeast is disrupted releasing intracellular proteins, enzymes and nucleic acids. The disrupted yeast is spray dried as a result of low pressure (0 psia to 200 psia) and higher temperatures in the spray drier (60° C. to 200° C.).
The spray-dried disrupted yeast cells in a powdered form are then contacted with SuperFluids (SFS) 290 in an extractor 280 to remove lipids in SFS 270. SFS used includes carbon dioxide, nitrous oxide, propane, alkanes and fluorocarbons. A preferred SFS is carbon dioxide. Pressures range from 500 psig to 5,000 psig. A preferred pressure is 3,000 psig. Temperatures range from 10° C. to 60° C. A preferred temperature is 40° C.
The delipidated, spray-dried disrupted yeast cells in a powdered form are then expanded through decompression valve 300. Rapid expansion of the SFS causes explosive disruption of the yeast cell walls producing yeast cell wall nanoparticles 310.
As a final process step 320, enzymatic cleavage of yeast cell nanoparticles produces Beta-glucans, chitins and mannans fractions. Hydrolysis, which may be acid-based, may also be performed on the cell nanoparticles to produce Beta-glucans, chitins, and mannans fractions. The fractionated yeast products 330 are produced in the final step of the process. The final process step can consist of a combination of enzymatic hydrolysis and acid hydrolysis.
The apparatus of shown in
A slurry conduit 80 for introducing a slurry of yeast cells into the mixing chamber 70 communicates with the inlet end 72. A high-pressure slurry pump 82 is connected to the slurry conduit 80 for pumping the slurry of cells under pressure into the mixing chamber 70. A solvent conduit 84 is in fluid communication with the slurry conduit 80 downstream of the slurry pump 82. A compressor 86 is provided along the solvent conduit 84 for raising the pressure of the solvent 22 and of the mixture within the mixing chamber 70 to critical pressures and above. A discharge conduit 88 leads from the outlet end 74 of the mixing chamber 70 to a blow-down chamber 90. A back pressure regulator or valve 87 is placed along the discharge conduit 88 between the mixing chamber 70 and the blow-down chamber 90 for continuously releasing the pressure on the slurry of cells exiting from the mixing chamber 70.
The blow-down chamber 90 is constructed and arranged to allow effective gravity separation of the solvent and the disrupted yeast slurry. In the embodiment shown, the lower end of the blow-down chamber 90 is funnel-shaped for collecting the disrupted cells. At the bottom of the funnel is an exit port 91. A liquid level control valve 92 is attached at the bottom exit port of the blow-down chamber 90 for controlling the liquid level within the blow-down chamber. Material may be collected at this port 91 or recycled via slurry recycle conduit 94 to the slurry conduit 80 upstream of the slurry pump 82.
A solvent recycle conduit 96 fluidly connects the upper exit of the blow-down chamber 90 to the solvent conduit 84, upstream of the compressor 86. Another back-pressure regulator 93 is located on the solvent recycle conduit 96 for controlling the pressure within the blow-down chamber 90.
Heat exchangers 98 are located just downstream of the solvent compressor 86 and the slurry pump 82 to regulate the temperature of solvent leaving the compressor. The temperature control loop 78 also controls the heat exchangers 98.
In operation, yeast cell slurry 54 may be fed directly from fermenters or centrifuges into the apparatus of
The pressure of the blow-down chamber may be maintained at pressures ranging from atmospheric to that of the mixing chamber. For a dominant coloration or permeability improvement mechanism, the pressure in the blow-down chamber 90 may be maintained at pressures relatively close to the operating pressures of the mixing chamber 70.
The continuous flow apparatus also may include a soaking chamber between the mixing chamber 70 and the blow-down chamber 90. Such a soaking chamber 100 will allow for a longer exposure time between the SFS solvent and the yeast cells; the soaking chamber may also accommodate mechanical mixers 102 to further facilitate the saturation of each yeast cell with SFS solvent. The soaking chamber can be bypassed by allowing the mixture of supercritical fluid and yeast slurry to flow directly from the mixing chamber 70 to blowdown chamber 90 via bypass loop 105.
For the process scribe in connection with
The effect of pressure on the supercritical disruption of Baker's yeast using N2O was also tested. The temperature and recirculation time were fixed at 40° C. and 25 minutes respectively. Pressure was varied from about 1,100 psig to 4,800 psig. As pressure increased, the recovery of nucleic acids and protein also increased. However, the relationship was more linear than that for E. coli, indicating that higher pressures may result in even higher recovery efficiencies.
The present invention utilizes SuperFluids to fractionate cellular biomass materials in two steps. In the first step, the biomass is disrupted by exposure to the critical fluid. It is hypothesized that this disruption involves at least two mechanisms, the first being liberation of cell envelope constituents to cause cell envelope permeability. The cell envelope constituents are not necessarily solvated in the critical fluid, i.e., they may remain in the phase containing the biomass, but in any case lose their structural association with the cell. The resulting permeability of the cell envelope makes certain contents of the cell accessible to be extracted in subsequent steps.
The second mechanism of disruption involves an explosive phenomenon due to the expanding SFS aka critical fluid upon depressurization of the biomass. In the latter case, rapid decompression is sometimes desirable. Larger systems may require longer to decompress than smaller systems. In the former case, decompression is not required to provide the desired disruption.
The nature of the biomass determines the relative importance of the two disruption mechanisms in any given application. During this first disruption step, an extract fraction may optionally be collected from the critical fluid contacting the biomass. In the second step of the fractionation, the disrupted biomass is subjected to a multiplicity of critical fluid extraction steps, the steps being characterized in that different solvation conditions are used in each. Thus, fractionation of the biomass is effected. As mentioned, critical fluid solvation properties may be varied by adjusting pressure, temperature, or modifier concentration. These parameters may be adjusted individually or in combination. Solvation conditions may also be varied through the use of different modifiers in a single fractionation procedure, although this would not typically be advantageous.
Preferably, each subsequent critical fluid is altered to change the solvation properties of the extracting fluid, so that each step can recover a different spectrum of compounds. The solvation properties of critical fluids can be altered by changing the temperature or pressure of the fluid. By way of example, a preferred temperature and pressure for a critical fluid comprising carbon dioxide is a temperature in the range of 10° C. to 60° C. and a pressure in the range of 500 psig to 5,000 psig.
Preferred critical fluids comprise carbon dioxide, nitrous oxide, ethylene, ethane, propane and freons. The fluid may also contain modifiers. Preferred modifiers are methanol, ethanol, propanol, butanol, methylene chloride, ethyl acetate and acetone.
A preferred modifier includes methanol. In one preferred embodiment, each subsequent extraction employs a larger concentration of methanol. Thus, the plurality of critical fluids becomes increasingly more hydrophilic. The first extraction step tends to remove lipophilic compounds while the last extraction step tends to remove hydrophilic compounds. Removal of the lipophilic materials allows the next more hydrophilic critical fluid to have access to more hydrophilic compounds trapped in cellular structures. Preferred methanol concentration ranges for a first extraction step on disrupted biomass, based on carbon dioxide at a pressure of 3000 psig and a temperature of 40° C., are 0-5 volume %. For the same temperature and pressure, 5-10 volume % methanol is preferred for a second extraction step; 10-20 volume % methanol is preferred for a third extraction step; 20-30 volume % methanol is preferred for a fourth extraction step; 30-50 volume % methanol is preferred for a fifth extraction step.
The combination of disruption and extraction with critical fluids produces larger numbers of fractions exhibiting biological activity than corresponding fractions derived from conventional organic solvent extractions. The use of critical fluids allows for easy removal of much of the solvent by mere depressurization. Use of a single apparatus to perform both the disruption and extraction steps minimizes labor and increases efficiency. Indeed, the entire process can be readily automated. The use of critical fluids allows the extraction conditions to be readily varied by temperature, pressure, or modifier solvents. Use of critical fluids for both the disruption and extraction simplifies the procedure and minimizes equipment needs, processing time, potential for contamination, and loss of yield. These and other features and advantages will be readily apparent from the drawing and detailed discussion which follow.
An alternative embodiment for lipid extraction is shown in
After loading a cartridge on the cartridge holder, the disruption/extraction procedure was commenced. The system was brought to 3000 psig and 40° C., and extracted for 10 minutes with pure CO2. This fraction was collected in methanol in a glass vial, numbered 4 in
With reference to
Critical fluid contained in cylinder 1 is supplied through line 2 and valve 4 to high pressure pump 3. With valve 12 closed and valve 5 open, high pressure pump 3 pressurizes line 7, chamber 8, and line 11. Pressure is indicated by pressure transducer 6. Once chamber 8 has been pressurized, the yeast cell wall and critical fluid are allowed a certain amount of contact time. After the desired contact time, valve 12 is quickly opened, e.g., in less than about 1 second, causing rapid depressurization of critical fluid with entrained yeast cell wall into the depressurization receptacle 15.
Depressurization may be carried out through a nozzle device 14, of which many designs are available. Some nozzle designs include impingement surfaces that increase mechanical shear by deflecting the discharging material.
The depressurization receptacle 15 is substantially larger than the contact chamber and operates at only a low pressure. It may be open to the atmosphere via a filter, which would trap any potentially escaping particles, although this is not shown in the figure. Alternatively, depressurization receptacle 15 may be a flexible container such as a plastic bag. After depressurization, yeast cell wall nanoparticles are collected from the depressurization receptacle 15 for analysis.
The general operation of the equipment was as described in the explanation of
It is intended that the subject matter contained in the preceding description be intended in an illustrative rather than a limiting sense.
This nonprovisional patent application claims priority to U.S. provisional application Ser. No. 63,051,079 filed on Jul. 13, 2020, the contents of which is incorporated by reference in its entirety.
Number | Date | Country | |
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63051079 | Jul 2020 | US |