Classifications

• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B02—CRUSHING, PULVERISING, OR DISINTEGRATING; PREPARATORY TREATMENT OF GRAIN FOR MILLING
  • B02C—CRUSHING, PULVERISING, OR DISINTEGRATING IN GENERAL; MILLING GRAIN
  • B02C11/00—Other auxiliary devices or accessories specially adapted for grain mills
  • B02C11/08—Cooling, heating, ventilating, conditioning with respect to temperature or water content
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B02—CRUSHING, PULVERISING, OR DISINTEGRATING; PREPARATORY TREATMENT OF GRAIN FOR MILLING
  • B02C—CRUSHING, PULVERISING, OR DISINTEGRATING IN GENERAL; MILLING GRAIN
  • B02C19/00—Other disintegrating devices or methods
  • B02C19/18—Use of auxiliary physical effects, e.g. ultrasonics, irradiation, for disintegrating
  • B02C19/186—Use of cold or heat for disintegrating
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D11/00—Solvent extraction
  • B01D11/02—Solvent extraction of solids
  • B01D11/0203—Solvent extraction of solids with a supercritical fluid

Definitions

• the physical form of feed i.e., the particle size and the attendant ratio of surface area to volume and volume to mass (specific volume) usually has a great impact on the efficiency of the process.
• This efficiency can be expressed in terms of using less solvent or energy, faster processing rate, higher percent recovery, and in many cases, higher quality of the final products.
• SCF supercritical fluids
• the greater the area of the solute exposed to solvent the more efficient the process is.
• freeze drying the heat flux and the rate of sublimation is directly proportional to the subject area of the material under process.
• Disruption of membranes, and maximum exposure can be obtained by treating plant or animal materials at or below their brittleness temperature. This is defined herein as the temperature below which a frozen material fractures into small particles when stress is applied thereto.
• animal or plant derived materials i.e., tissues, organs, cells and organelles for separation processes such as extraction and leaching via aqueous solvents, organic solvents, SCF, and/or application of thermal energy
• the solutes usually contents of the cells, protoplasm and/or membranes
• the cell membrane should be ruptured. The greater the ratio of surface area to volume of such animal derived solutes, the more efficient the process is.
• adipose tissues and omentum cannot be homogenized (ground) at room temperature, refrigerator temperature, or even higher freezing temperatures.
• the high plasticity of these tissues is not compatible with grinding processes.
• phosphate buffered saline (PBS) or water is added as a "filler" to animal fatty tissues in order to make the homogenization possible.
• PBS phosphate buffered saline
• the tissue cannot be disintegrated by mere homogenization.
• Addition of aqueous phase requires its subsequent removal from the system by a time consuming or energy consuming step such as centrifugation or freeze drying.
• Yet another object of this invention is to extract materials from animal and plant which have been treated at brittleness temperatures or below to obtain desired substances therefrom.
• Yet a further object of the invention is to provide a method of separation such as drying, freeze drying, leaching and extraction using aqueous and organic solvents and/or supercritical fluids (SCFs) in connection with materials fractured at or below their brittleness temperatures.
• aqueous and organic solvents and/or supercritical fluids SCFs
• the larger tissues should be cut into relatively small pieces (e.g., 1-5 grams). This will improve the freezing rate of tissues and facilitate their later handling during the cryogrinding step or steps. Attempts should be made to minimize pretreatment time and tissues should become frozen without undue delay.
• This step is based on freezing the pieces of the desired tissue and lowering its temperature to an emperically critical "Brittleness Temperature" which converts the unbreakable, viscous, and sticky (e.g. fatty tissue) materials into an extremely brittle and fragile substance.
• Brittleness temperature depends primarily on the species and composition of the tissue (water, lipids, proteins, carbohydrates, minerals) and therefore its thermal properties.
• the rate of crystallization (i.e., nucleation and crystal growth) of water will affect the size of crystals. Slow rate of crystallization results in formation of large extracellular water crystals which may cause some rupture of cell membranes, but such effects and even repeated freeze-thaw process have negligible effects in comparison to cell rupture by cryogrinding. It is preferred, however, to keep the physical structure of the tissue cells up to the cryogrinding step, intact. This means that, all other factors being equal, the fastest possible freezing rate should be employed.
• the initial volume increase of the tissue Pure water at 0°C expands approximately 9% when transformed into ice at the same temperature. Most tissues also expand on freezing but to a lesser extent than pure water.
• Freezing can be accomplished via various methods. including;
• Air Freezing a) Blast freezing b) Fluidized-bed freezing
• Liquid-immersion Freezing 4. Cryogenic Freezing (freezant undergoes a change of state): a) Liquid Nitrogen (LN 2 ), -196°C (77K) b) Subliming Carbon dioxide (dry ice), -79°C (194K) c) CCl 2 F 2 (refrigerant -12), -30°C (243K) Cryogenic freezing with LN 2 , among all above methods, is the most desirable method for the following reasons:
• Liquid nitrogen is a safe, non-toxic, and non-flammable cryogenic medium which is universally used in food, pharmaceutical, and other industries.
• tissue powder Upon obtaining the brittle pieces of tissues, they may be transferred to any size reduction equipment such as Waring blender and homogenized (ground) for the desired length of time, i.e., a few minutes at 22,000 RPM. At larger scales, roll mills with both attrition and impact grinding could be used. It is important that the tissue be kept at or below its brittle temperature throughout the grinding process. Grinding below the brittleness temperature is required in order to produce the necessary small particle size. For this purpose one could occasionally add, if needed, quantities of LN 2 to the size-reduction equipment, provided that there is a vent to allow exhaustion of the nitrogen vapor generated during the process.
• tissue powder Upon completion of the process, an extremely fine, and free-flowing (non-sticky) cryoground tissue ("tissue powder”) results. The resultant powder obviously may contain some granular lumps as well as fine indiscrete particles.
• density of omentum powder is 0.44 ( ⁇ 5%) g/ml which is almost half of the density of lipids extracted from omentum.
• tissue powder while extremely fine, is not "uniform" with regard to size. Consequently, if uniformity in a particular size range is desired for the following steps, tissue powder should be sieved at or below its brittleness temperature. For this purpose, one may use stacked stainless steel standard sieves (Table 1; AOAC, 1984).
• LN 2 is poured over the top sieve to cool the whole system down to LN 2 temperature.
• an appropriate amount of cryoground tissue powder is placed in the top sieve and the entire stack is subjected to a uniform vibrating process (shaker) for a few minutes. It is important that the tissue powder be kept at or below the brittle temperature, by occasional addition of LN 2 throughout the cryosieving process.
• tissue powder is recovered from the top of each individual sieve and can be used individually or in a desired combination of particle sizes. Oversize particles can be recycled for further cryogrinding. Distribution of particle size can be obtained from weighing of material on each sieve.
• some chemical changes e.g., oxidation of unsaturated lipids, especially because of tremendous surface area generated by cryogrinding; insolubilization or destabilization of proteins; and degradation of pigments and vitamins and other biomolecules
• Reduction of freezer temperature will cause decline of the rates of the above reactions. Consequently, for longer storage times, it is recommended that the final tissue powder be stored at -40°C, under vacuum or inert gas, and in the dark, (to prevent any possible photo-catalytic reactions).
• Preliminary evaluation shows that various tissue powders stored under the above conditions for up to 2 months, did not show any physical changes (texture, color, odor, etc.) in the product.
• tissue powder To use the uniform cryoground product, one should desirably "thaw" the tissue powder. Since thawing of non-fluid tissues is inherently slower than freezing, when comparable temperature differentials are employed (due to different thermal properties of ice vs. water). Hence, tissue powders may be subject to damage by chemical or physical (and less microbial or enzymatic) means. In light of these considerations, one skilled in the art will recognize that the thawing process must be carefully considered.
• the process is straight forward, non-complicated, effective, fast, and clean with minimum loss. Since no medium is added for homogenization, there is no need for an extra step (e.g., centrifugation) for removal of any medium.
• the process is technology adaptable.
• cryoground and cryosieved tissues are free-flowing powders, their handling (e.g., weighing, transferring, mixing, pouring, etc.) in laboratory and/or plant is very easy.
• tissue powders due to their extreme homogeniety, may be used as reliable common sources for comparative analytical and preparative research and development studies.
• cryogrinding was applied to porcine omentum and a pinkish "omentum powder" was obtained. Upon cryosieving, the best uniform fraction of omentum powder appeared to be in the range of 150 to 600 um.
• cryogrinding was applied to porcine brain and a white "brain powder" was obtained. Upon cryosieving, the best uniform fraction of brain powder appeared to be in the range of 300 um to 1.18 mm.
• pancreas powder was obtained.
• the best uniform fraction of pancreas powder appeared to be in the range of 150 um to 1.18 mm.
• liver powder was obtained.
• the best uniform fraction of liver powder appeared to be in the range of 300 um to 1.18 mm.
• kidney powder was obtained. Upon cryosieving, the best uniform fraction of kidney powder appeared to be in the range of 300 um to 1.18 mm.
• cryogrinding was applied to porcine spleen and a "spleen powder" was obtained.
• the best uniform fraction of spleen powder appeared to be in the range of 300 um to 1.18 mm.
• cryogrinding was applied to porcine blood and a "blood powder" was obtained. Upon cryosieving, the best uniform fraction of blood powder appeared to be in the range of 150 to 300 um.
• brain spinal chord, spinal fluid, appendages
• peripheral nervous system tissues and organs cranial nerves, spinal nerves, etc.
• lymphatic system tissues and organs lymphatic system tissues and organs (lymph nodes, spleen, thymus); respiratory system tissues and organs (upper respiratory tract, lungs); digestive system tissues and organs (including mouth, teeth, tongue, salivary glands, pharynx, esophagus, peritoneum, stomach, small and large intestine, liver, gall bladder, pancreas); skeletal tissue and organs (axial and appendicular skeleton, bone marrow); muscles (smooth and skeletal); endothelial and epithelial tissue; membranes, omentum, and cartiligenous tissues
• tendons, ligaments, joints tendons, ligaments, joints
• sensory organs eyes, ear, nose, tongue
• endocrine or other glandular tissue thyroid gland, parathyroid gland, pituitary gland, adrenal gland
• urinary tissue and organs kidneys, ureters, urinary bladder, urethra
• reproductive organs and tissues testes, ovaries, etc.
• adipose tissues such as is contained in subcutaneous and internal organs, as well as biological exudates, such as feces, urine, sweat, semen, milk, and so forth, are used. In each case such processing conditions are chosen which optimizes the physical and rheological characteristics of the desired powder.
• Chloroform/Methanol Extraction of Omentum Powder 500 g. uniform porcine omentum powder was warmed up to room temperature and extracted with 10 times chloroform/methanol (2:1, v/v) in a glass blender (22,000 RPM, 30 seconds). The solvent extract was centrifuged (2,000 RPM, 20 minutes) and subjected to rotary evaporation (under vacuum, 37°C) until dryness, i.e., neither any solvent condensation occurs, nor any solvent odor is present. A whitish chloroform/methanol fraction (CMFr) weighing 388 g. (i.e., 77.6%) was obtained. The CMFr could be further subjected to a hexane/ethanol fractionation.
• CMFr chloroform/methanol fraction
• cryogrinding causes production of particles with smaller sizes. This may improve the oil recovery, when compared to equally treated, but flaked or R.T. ground soy flour.
• 250 g. of the resultant cryoground soy flour (from 600 um sieve) was extracted with 15 times hexane at room temperature.
• the resultant cloudy solution was centrifuged at 2000 RPM for 20 minutes. Upon centrifugation, the clear yellowish supernatant was rotary evaporated at 37°C under vacuum. Total recovery was 24.3 g. (i.e., 9.7%).
• the materials which can be extracted using the processes described herein include, but are not limited to, complex lipids, such as acylglycerols, phosphoglycerides, sphingolipids, gangliosides and waxes; simple lipids, such as terpenes, pigments, steroids and their alcohols (sterols), prostaglandins, and so forth.
• complex lipids such as acylglycerols, phosphoglycerides, sphingolipids, gangliosides and waxes
• simple lipids such as terpenes, pigments, steroids and their alcohols (sterols), prostaglandins, and so forth.
• Glycolipids, lipoproteins, membrane supramolecular complexes, and their metabolic intermediates be they catabolic or anabolic, and metabolic products of these molecules, as well as molecules which behave in a fashion similar to lipids, may be obtained in a fashion similar to that given in the examples, supra.
• Proteinaceous substances such as amino acid containing substances (including non-protein amino acids), oligopeptides, peptides, polypeptides, hormones, proteins, enzymes, antibodies, fractions and components of these, as well as metabolic intermediaries and products may be obtained. While the choice of temperatures, solvents, SCFs and reaction parameters will vary, depending upon the substance to be extracted, one skilled in the art will be able to determine which reagents and conditions to use. Saccharides, including mono-, di-, oligo- and polysaccharides, as well as glycoproteins may be extracted in this way as well. Again, metabolic intermediates and products can be obtained as well.
• nucleotide family of molecules including purines and pyrimidines, and any molecules containing nucleic acid bases, nucleosides (ribonucleosides and deoxyribonucleosides), nucleic acids, supramolecular complexes of nucleic acids and proteins, viruses, and so forth as well as their intermediates and products, metaolic products may also be obtained.
• materials not grouped into one of the "families” listed supra may be obtained. These include all fat and/or water soluble vitamins, flavors, flavor potentiators, their intermediates, both catabolic and anabolic, and products as well.
• the desired material may also be treated with one or any combination of more than one of the following methods of treating the samples:
• the samples need not be treated prior to cryogrinding, but may, e.g., be treated after the preparation of tissue powder.
• Supercritical fluid extraction may be accomplished with many different gases, including those listed in the following Table II.

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• 1986
  • 1986-12-16 IL IL81000A patent/IL81000A0/xx unknown
  • 1986-12-17 AU AU68392/87A patent/AU584780B2/en not_active Ceased
  • 1986-12-17 JP JP62500408A patent/JPS63502090A/ja active Pending
  • 1986-12-17 WO PCT/US1986/002741 patent/WO1987003951A1/en not_active Application Discontinuation
  • 1986-12-17 EP EP19870900565 patent/EP0252124A4/de not_active Withdrawn
  • 1986-12-17 KR KR870700751A patent/KR880700688A/ko not_active Application Discontinuation
  • 1986-12-19 ZA ZA869569A patent/ZA869569B/xx unknown
  • 1986-12-19 NZ NZ218720A patent/NZ218720A/xx unknown
• 1987
  • 1987-08-19 DK DK432387A patent/DK432387D0/da not_active Application Discontinuation
  • 1987-08-20 FI FI873598A patent/FI873598A0/fi not_active IP Right Cessation

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