WO2016090477A1 - Sterile methods and products - Google Patents

Sterile methods and products Download PDF

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Publication number
WO2016090477A1
WO2016090477A1 PCT/CA2015/051288 CA2015051288W WO2016090477A1 WO 2016090477 A1 WO2016090477 A1 WO 2016090477A1 CA 2015051288 W CA2015051288 W CA 2015051288W WO 2016090477 A1 WO2016090477 A1 WO 2016090477A1
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Prior art keywords
algae
products
fatty acid
copper
extract
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PCT/CA2015/051288
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French (fr)
Inventor
Hajaniriana Eric ANDRIANASOLO
Kristen MULLER
Brendan MCCONKEY
Sarah RUFFELL
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Algae Dynamics Corp.
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Publication of WO2016090477A1 publication Critical patent/WO2016090477A1/en

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Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
    • A23B9/00—Preservation of edible seeds, e.g. cereals
    • A23B9/16—Preserving with chemicals
    • A23B9/24—Preserving with chemicals in the form of liquids or solids
    • A23B9/30—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/02—Saturated carboxylic acids or thio analogues thereof; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/03—Algae
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
    • A23B9/00—Preservation of edible seeds, e.g. cereals
    • A23B9/16—Preserving with chemicals
    • A23B9/24—Preserving with chemicals in the form of liquids or solids
    • A23B9/26—Organic compounds; Microorganisms; Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/42—Preservation of non-alcoholic beverages
    • A23L2/44—Preservation of non-alcoholic beverages by adding preservatives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
    • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
    • A23L3/3463—Organic compounds; Microorganisms; Enzymes
    • A23L3/3472—Compounds of undetermined constitution obtained from animals or plants
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
    • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
    • A23L3/3463—Organic compounds; Microorganisms; Enzymes
    • A23L3/3481—Organic compounds containing oxygen
    • A23L3/3508—Organic compounds containing oxygen containing carboxyl groups
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
    • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
    • A23L3/358—Inorganic compounds

Definitions

  • the invention relates to the field of food sterilization.
  • Pasteurization is a heating process that reduces the number of viable pathogens to give the product shelf life.
  • Sterilization is a heating process that kills all micro-organisms in the product.
  • Forming one aspect of the invention is method of sterilization and product preparation, the method comprising the step of mixing the product with (i) algae or an algae derivative; and (ii) a disinfectant adapted for Agrobacterium tumefaciens.
  • the product can be one of grits, polenta, corn meal, corn flour and orange juice.
  • the algae can be Phaeodactylum tricornutum.
  • the algae can be one of Rhodella maculata (Rhodophyta), Boekelovia hooglandii (Heteromonyphyta), Goniochloris sculpta
  • Heteromony sp. Chlo della simplex (Heteromony sp.), Ascophyllum sp., Fucus sp., Himanlhalia sp., Undaria sp., and Laminaria sp.
  • the disinfectant can be one of : Ti0 2 ; copper; copper-containing sulfur doped Ti0 2 ; and copper, silver and nickel-enriched Ti0 2
  • the algae derivative can be an extract from algae containing fatty acid, the fatty acid ranging from C12 to C22.
  • the algae derivative can be an extract from algae having added with purified siliceous frustules cell wall.
  • the algae derivative can be an extract added with purified and engineered siliceous frustules cell wall to contain Ti(OH) 4 in situ.
  • the disinfectant can be Ti0 2 in the form of anatase or nanocrystalline.
  • the product can be selected from corn meal, grits, polenta and corn flour
  • the resultant mixture can have 90-95 wt % product.
  • the resultant mixture can have 4-10 wt% algae.
  • the resultant mixture can have up to 1% disinfectant.
  • the extract can be an omega 4 polyunsaturated fatty acid isolated from Phaeodactylum tricornutum.
  • a product is mixed with (i) algae or an algae derivative; and (ii) a disinfectant adapted for Agrobacterium tumefaciens.
  • the resultant composition is submitted to be relatively sterile, relatively shelf-stable and relatively appetizing, all suitable at least for use as animal feed.
  • Phaeodactylum tricornutum has useful antibiotic properties against most common bacteria, notably excluding Agrobacterium tumefaciens.
  • Example 1 Extraction and Isolation Procedures. Laboratory Cell Culturing.
  • Phaeodactylum tricornutum Bohlin clone Pt1 8.6 were obtained from the culture collection of the Provasoli- Guillard National Center for Marine Algae and Microbiota formerly named National Center for Culture of Marine Phytoplankton, Bigelow Laboratory for Ocean Sciences, West Boothbay Harbor, ME. Cultures were grown in f/2 medium made from 0.2 ⁇ filtered, autoclaved seawater supplemented with filter-sterilized vitamins and inorganic nutrients. Cultures were incubated at 18 °C under constant light (mean intensity 100 ⁇ m "2 s "1 ). Sterility was monitored by occasional inoculation into peptone-enriched media to check for bacterial growth.
  • Diatom cells were harvested by centrifugation for approximately 15 min at 4000g, and pellets were rapidly frozen in liquid N2 and stored at -80 °C before further workup in the laboratory.
  • the material 40 g was extracted three times with MeOH to give a polar crude organic extract (420 mg).
  • a portion of this extract (15 mg) was tested for antimicrobial or apoptosis induction.
  • the crude organic extract was found active and subjected to fractionation using a solid-phase extraction cartridge (normal-phase silica) to give four fractions, F1 to F4, using hexane, CH2CI2, EtOAc, and MeOH as an increasingly hydrophilic solvent system series.
  • This fraction was further chromatographed on analytical RP HPLC (Phenomenex luna C18, 250 * 4.60 mm) using isocratic elution with 100% MeOH (flow rate 1 mL/min) to yield several pure fatty acids (10 mg average weight each), readily used for sterile compositions.
  • Example 3 Siliceous frustules cell wall isolation and purification
  • the purification of the cell wall can be done by heating in an oven. Siliceous substances and inorganic substances will not be calcinated but fatty acids and other organic substances will be calcinated and eliminated around 350-500 degree C, or preferably at 600 degree C. The white and grayish substance remaining after calcination was readily used for sterile compositions.
  • the medium composition can be adjusted during the culture of Phaeodactylum tricornutum and add titanium salt or titanium hydroxide on the media.
  • the organism will incorporate titanium hydroxide or titanium oxide into the structure of frustules cell wall, and the purification of the cell wall obtained by this method can also be done with the same process described above.
  • Certain fatty acids [10:0, 14:0, 16:0, 17:0, 18:0, 20:0, 22:1 (n-9), 23:0, 24:0, and 24:1 (n-9)] were heated to 50 °C to improve solubility in alcohol.
  • Seven-millimeter-diameter paper discs [Becton, Dickinson and Company (Canada)] were loaded with appropriate volumes of stock solution to result in a disc containing 2000, 800, 250, or 25 ig of fatty acid.
  • Tetracycline was used as a positive control and as a basis for comparison to determine how effective the fatty acids are relative to a typical dose of a common antibiotic (Heman-Ackah 1976).
  • a tetracycline stock solution of 50 mg mL "1 was diluted with sterile water and used to prepare 5- and 30 ⁇ g discs of tetracycline, the latter of which is the Clinical and Laboratory Standards Institute (CLSI) standard (Clinical and Laboratory Standards Institute 2006). Ethanol-soaked discs were additionally prepared as solvent controls. Disk diffusion was selected as the test method to be consistent with standard methodology used by the CLSI for evaluating antibiotic effectiveness against bacteria (Sader et al. 2007). Spread plating was used to disperse the bacteria on plates. Loaded discs and bacterial plates were placed in a biosafety cabinet at room temperature until dry; then, loaded discs were added to the bacterial plates.
  • CLSI Clinical and Laboratory Standards Institute
  • a scale up culture is needed.
  • a novel method of cultivating algae was used which combines the positive features of open pond systems with those of enclosed photobioreactor algae production systems.
  • the system produces a continuous supply of ultra-pure algae biomass in high volumes.
  • the design enables full control of all cultivation parameters allowing to achieve optimum growing conditions for any algae species.
  • a unique C0 2 delivery system is used that enhances delivery efficiency and minimizes C0 2 losses (GHG emissions) from the system.
  • strains were cultured from the system: Chlorella, Scenedesmus.
  • Algal oil was produced and the majority of the oils are fatty acids that were readily used for sterile compositions.
  • Example 6 Sterilization method using fatty acid isolated from cultured algae
  • Example 7 Sterilization method using fatty acid isolated from cultured algae
  • fatty acid ranging from C12 to C22 isolated from cultured algae Phaeodactylum tricornutum and produced as described in example 1 and 5 were mixed with 900g squeezed fresh orange. These products were mixed intensively in a mixer for 5min. The mixture was added with 10g of Titanium Oxide Nanoparticles (Ti0 2 , anatase, 99%, 15 nm) purchased from NanoAmor Europe. The mixture were left in open atmosphere at room temperature 25 °C for 1 day and then tested for sterility from common bacteria as described in example 4. The products were found to be free or at least 5-log reduction from the following bacteria: E. coli, S. aureus and specifically Agrobacterium tumefaciens.
  • Example 8 Sterilization method using fatty acid isolated from cultured algae
  • Phaeodactylum tricornutum and cuprous chloride Phaeodactylum tricornutum and cuprous chloride.
  • Example 9 Sterilization method using fatty acid isolated from cultured algae
  • Example 10 Sterilization method using cultured algae Phaeodactylum tricornutum
  • Example 11 Sterilization method using cultured algae Phaeodactylum tricornutum and Ti0 2 in the form of anatase or nanocrystalline
  • Example 12 Sterilization method using cultured algae Phaeodactylum tricornutum and Copper (Cu) Nanopowder APS: 70 nm.
  • Example 14 Sterilization method using extract of cultured algae Phaeodactylum tricornutum and engineered Siliceous frustules cell wall
  • Example 15 Sterilization method using extract of cultured algae Phaeodactylum tricornutum and Ti0 2 in the form of anatase or nanocrystalline
  • Example 16 Sterilization method using fatty acid isolated from cultured algae
  • fatty acid ranging from C12 to C22 isolated from cultured algae Phaeodactylum tricornutum and produced as described in example 1 and 5 were mixed with 900g squeezed fresh orange. These products were mixed intensively in a mixer for 5min. The mixture was added with 10g of engineered siliceous frustules cell wall as described in example 3. The mixture products were left in open atmosphere at room temperature 25 °C for 1 day and then tested for sterility from common bacteria as described in example 4. The products were found to be free or at least 5-log reduction from the following bacteria: E. coli, S. aureus and specifically Agrobacterium tumefaciens.
  • Example 17 Data reported under pH> 6 for sterilization method using fatty acid isolated from cultured algae Phaeodactylum tricornutum and Ti0 2 in the form of anatase or nanocrystalline
  • Example 18 Sterilization method using fatty acid isolated from cultured algae Phaeodactylum tricornutum and Nitrogen and sulfur co-doped Ti02 it is known that the overall efficiency of Ti0 2 under natural sunlight is limited to the UV-driven activity ( ⁇ ⁇ 400 nm), shifting the absorption spectrum of Ti0 2 towards the visible-light range is possible by incorporation of no-metallic dopants into its lattice. For this purpose example 7 was repeated with a synthetic Nitrogen and sulfur co-doped Ti0 2 .
  • fatty acid ranging from C12 to C22 isolated from cultured algae Phaeodactylum tricornutum and produced as described in example 1 and 5 were mixed with 900g squeezed fresh orange. These products were mixed intensively in a mixer for 5min. The mixture was added with 10g of Nitrogen and sulfur co-doped Ti0 2 synthesized from Titanium Oxide Nanoparticles and using manual grinding with thiourea and annealing at 400 °C (J.A. Rengifo-Herrera, C. Pulgarin / Solar Energy 84 (2010) 37-43). The mixture were left in open atmosphere at room temperature 25 °C for 1 day and then tested for sterility from common bacteria as described in example 4. The products were found to be free or at least 5-log reduction from the following bacteria: E. coli, S. aureus and specifically Agrobacterium tumefaciens.
  • Example 7 was repeated using polenta made by boiling cornmeal instead of squeezed fresh orange.
  • 90g of fatty acid ranging from C12 to C22 isolated from cultured algae Phaeodactylum tricornutum and produced as described in example 1 and 5 were mixed with 900g polenta. These products were mixed intensively in a mixer for 5min. The mixture was added with 10g of Titanium Oxide Nanoparticles (Ti0 2 , anatase, 99%, 15 nm) purchased from NanoAmor Europe.
  • Example 20 Sterilization method using cultured algae Phaeodactylum tricornutum and Ti0 2 in the form of anatase or nanocrystalline and polenta as food sample
  • the following variant of example was also experimented. 90g of cultured algae Phaeodactylum tricornutum produced as described in example 5 were ground with final particles size of 0.1 pm. These greenish products were mixed intensively with 900g of polenta made by boiling cornmeal. The yellow greenish mixture was added with 10g of Titanium Oxide Nanoparticles (Ti0 2 , anatase, 99%, 15 nm) purchased from NanoAmor Europe. The mixture products were left in open atmosphere at room temperature 25 °C for 1 day and then tested for sterility from common bacteria as described in example 4. The products were found to be free or at least 5-log reduction from the following bacteria: E. coli, S. aureus and specifically Agrobacterium tumefaciens.
  • Example 21 Time dependent growth of bacteria
  • fatty acid ranging from C12 to C22 isolated from cultured algae Phaeodactylum tricornutum and produced as described in example 1 and 5 were mixed with 900g squeezed fresh orange. These products were mixed intensively in a mixer for 5min. The mixture was added with 5g of Titanium Oxide Nanoparticles (Ti02, anatase, 99%, 15 nm) purchased from NanoAmor Europe and 0.05mg of Copper (Cu) Nanopowder APS (70 nm) purchased from US Research Nanomaterials, Inc.. The mixture were left in open atmosphere at room temperature 25 °C for 1 day and then tested for sterility from common bacteria as described in example 4. The products were found to be free or at least 5-log reduction from the following bacteria: E. coli, S. aureus and specifically Agrobacterium tumefaciens.
  • the main components are fatty acids, extract of algae or cultured algae and the disinfectants are Ti0 2 , copper in the form of nanoparticles or engineered Siliceous frustules cell wall.
  • the weight percentage ranges of all components depend on the form of the material to be sterilized.

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Abstract

Disclosed therein is a method for sterilizing a product and comprises mixing a product with (i) algae or with an algae derivative; and (ii) a disinfectant adapted for Agrobacterium tumefaciens. The product includes one of grits, polenta, corn meal, corn flour and orange juice. The algae is one of Rhodeella maculata, Boekelovia hooglandii, Goniochloris sculpta, Chloridella simplex, Ascophyllum sp., Fucus sp., Himanlhalia sp., Undaria sp., and Laminaria sp. The algae derivative is an extract form algae containing fatty acid, the fatty acid ranging from C12 to C22. The disinfectant includes one of TiO2, copper, copper-containing sulfur doped TiO2; and copper, silver and nickel-enriched TiO2.

Description

STERILE METHODS AND PRODUCTS
FIELD OF THE INVENTION
The invention relates to the field of food sterilization.
BACKGROUND OF THE INVENTION
It is known to heat goods such as foodstuffs to kill pathogens such as bacteria. Pasteurization is a heating process that reduces the number of viable pathogens to give the product shelf life. Sterilization is a heating process that kills all micro-organisms in the product.
Both processes are known to have usefulness, but heating products has an associated cost and heat can also adversely affect taste and quality of foodstuffs.
SUMMARY OF THE INVENTION
Forming one aspect of the invention is method of sterilization and product preparation, the method comprising the step of mixing the product with (i) algae or an algae derivative; and (ii) a disinfectant adapted for Agrobacterium tumefaciens.
According to another aspect of the invention, the product can be one of grits, polenta, corn meal, corn flour and orange juice.
According to another aspect of the invention, the algae can be Phaeodactylum tricornutum. According to another aspect of the invention, the algae can be one of Rhodella maculata (Rhodophyta), Boekelovia hooglandii (Heterokontophyta), Goniochloris sculpta
(Heterokontophyta), Chlo della simplex (Heterokontophyta), Ascophyllum sp., Fucus sp., Himanlhalia sp., Undaria sp., and Laminaria sp.
According to another aspect of the invention, the disinfectant can be one of : Ti02 ; copper; copper-containing sulfur doped Ti02 ; and copper, silver and nickel-enriched Ti02
According to another aspect of the invention, the algae derivative can be an extract from algae containing fatty acid, the fatty acid ranging from C12 to C22.
According to another aspect of the invention, the algae derivative can be an extract from algae having added with purified siliceous frustules cell wall.
According to another aspect of the invention, the algae derivative can be an extract added with purified and engineered siliceous frustules cell wall to contain Ti(OH)4 in situ.
According to another aspect of the invention, the disinfectant can be Ti02 in the form of anatase or nanocrystalline.
According to another aspect of the invention, the product can be selected from corn meal, grits, polenta and corn flour
According to another aspect of the invention, the resultant mixture can have 90-95 wt % product.
According to another aspect of the invention, the resultant mixture can have 4-10 wt% algae.
According to another aspect of the invention, the resultant mixture can have up to 1% disinfectant.
According to another aspect of the invention, the extract can be an omega 4 polyunsaturated fatty acid isolated from Phaeodactylum tricornutum.
DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENT
In a method according to an exemplary embodiment of the invention, a product is mixed with (i) algae or an algae derivative; and (ii) a disinfectant adapted for Agrobacterium tumefaciens.
Without intending to be bound by theory, the resultant composition is submitted to be relatively sterile, relatively shelf-stable and relatively appetizing, all suitable at least for use as animal feed.
In support of the above, experimental testing by the inventors has demonstrated that
Phaeodactylum tricornutum has useful antibiotic properties against most common bacteria, notably excluding Agrobacterium tumefaciens.
Table 1. The lowest effective fatty acid dose (pg) of the four tested dosages (2000 pg, 800 g, 250 g and 25 g) at 24 and 48 hours, resulting in bacterial growth inhibition of the three bacteria tested, E. coli, S. aureus and A. tumefaciens. (Ruffell et al., 2015)
Bacteria Escherichia Staphylococcus Agrobacterium coli aureus tumefaciens
Fatty Acids 24 h 48 h 24 h 48 h 24 h 48 h
10:0 Decanoic acid 2000 Nl* 800 800 Nl Nl
12:0 Laurie acid Nl Nl 2000 Nl Nl Nl
14:0 Myristic acid Nl Nl Nl Nl Nl Nl
16:0 Palmitic acid Nl Nl Nl Nl Nl Nl
17:0 Heptadecanoic acid ffi dtt s eecvec Nl Nl Nl Nl Nl Nl
18:0 Stearic acid Nl Nl Nl Nl Nl Nl
20:0 Arachidic acid Nl Nl Nl Nl Nl Nl
23:0 Tricosanoic acid Nl Nl Nl Nl Nl Nl
24:0 Lignoceric acid Nl Nl Nl Nl Nl Nl
14:1 Myristoleic acid Nl Nl 2000 2000 Nl Nl
16:1 Palmitoleic acid Nl Nl 250 800 Nl Nl o
8: 1 (n- Vaccenic acid ^3 Nl Nl Nl Nl Nl Nl8: 1 (n- Oleic acid CO Nl Nl Nl Nl Nl Nl0: 1 (n- 1 1 -Eicosenoic acid Nl Nl Nl Nl Nl Nl2: 1 (n- Erucic acid Nl Nl Nl Nl Nl Nl4: 1 (n- Nervonic acid Nl Nl Nl Nl Nl Nl8:2 (n- Linoleic acid Nl Nl 800 800 Nl Nl ω
8:3 (n- Gamma-linolenic acid Nl Nl 250 250 Nl Nl o
0:2 (n- 1 1-14 Eicosadienoic acid Nl Nl Nl Nl Nl Nl0:3 (n- Homo-gamma-linolenic Nl Nl 800 800 Nl Nl0:4 (n- Arachidonic acidb Nl Nl 250 250 Nl Nl2:2 (n- 13-16 Docosadienoic acid 800 Nl Nl Nl Nl Nl2:4 (n- Docosatetraenoic acid Nl Nl 250 2000 Nl Nl2:5 (n- Docosapentaenoic acid n6 Nl Nl 250 800 Nl Nl8:3 (n- Linolenic acid 25 Nl 250 250 Nl Nl0:3 (n- 1 1 ,14,17- Eicosatrienoic Nl Nl 2000 Nl Nl Nl0:5 (n- Eicosapentaenoic acid Nl Nl 250 250 Nl Nl2:5 (n- Docosapentaenoic acid n3 Nl Nl 800 2000 Nl Nl2:6 (n- Docosahexaenoic acid Nl Nl 800 800 Nl Nl
Control (ethanol) 0 0 0 0 0 0 a 90% purity; everything else is 99% purity b Nl = No Inhibition Table 2. The fatty acid response of the five test algae to two different light cycles, 24:0 (24 hours light: 0 hours dark) and 17:7 (17 hours light: 7 hours dark). (Ruffell et al., 2015)
Light Fatty acid (mg/g) Total % of
_ Antibiotic .
Duration p» Total
C10:0 C16:l C18:3(n-6) C20:4(n-6) C22:2(n-6) tA
FAa
Boekelovia 24 h 1.68E-03± 28.7 ± 0.226 ± 0.714 ± 2.20E-02 ± 29.6 ± 23.6
1.22E-03 2.53A 3.68E-02Ab 1.47E-01A 1.07E-02 2.54A
hooglandii
17h l .29E-02 ± 20.5 ± 0.108± 0.189 ± 7.70E-02 ± 20.9 ±3.05 23.8
1.49E-02A 2.96 2.05E-02 8.57E-02 7.76E-02
Phaeodactylum 24 h 4.10E-03 ± 63.1 ± 0.440 ± 0.525 ± 3.70E-02± 64.1 ± 41.8
1.77E-03 5.25A 2.51E-02 4.31E-02 9.21E-03 5.23 A
tricornutum
17h 1.72E-02± 34.1 ± 0.488 ± 1.03 ± 3.30E-02± 35.7 ±4.82 39.1
9.40E-03A 4.53 8.99E-02 2.43E-01 1.66E-02
Goniochloris 24 h 2.66E-03 ± 0.578 ± 0.247 ± 3.65 ± 7.18E-02± 4.55 ± 8.7
2.89E-03 0.128 5.47E-02A 1.04 A 1.89E-02 1.23A
sculpta
17h 4.99E-03 ± 0.518 ± 0.100± 1.59 ± 1.12E-01 ± 2.33 ± 5.1
4.40E-03 7.53E-02 1.30E-02 4.07E-01 2.05E-02 4.71E-01
Rhodella maculata 24 h l.l2E-02± 0.961 ± 0.130± 0.01 ± 7.66 ± 1.08 8.77 ± 1.20 29.7
5.811E-03A 9.49E-02 1.69E-02 2.18E-03
17h 3.32E-03 ± 1.89 ± 0.172 ± 7.81 ± 1.36 5.10E-02 ± 9.93 ± 1.12 20.0
3.90E-03 5.22E- 4.04E-02 2.71E-02A
01 A
Chloridella 24 h 5.02E-02 ± 41.3± 0.315 ± 4.32 ± 1.80E-02± 46.0 ± 34.2
5.89E-03A 1.04 A 2.55E-02A 1.56E-01A 1.98E-03 1.09A
simplex
17h 2.31E-03 ± 36.6 ± 0.179± .08 ± 4.60E-02 ± 39.9 ±2.23 28.9
1.80E-03 1.93 2.21E-02 2.87E-01 1.55E-02A
a Total Antibiotic FAs as % of total FA; b A, B, and C represent statistical significance (refer to Statistical Analysis for details
The breadth and scope of the invention is made evident by the examples which follow
Example 1 : Extraction and Isolation Procedures. Laboratory Cell Culturing.
Axenic cultures of Phaeodactylum tricornutum Bohlin clone Pt1 8.6 (CCMP2561) were obtained from the culture collection of the Provasoli- Guillard National Center for Marine Algae and Microbiota formerly named National Center for Culture of Marine Phytoplankton, Bigelow Laboratory for Ocean Sciences, West Boothbay Harbor, ME. Cultures were grown in f/2 medium made from 0.2 μηι filtered, autoclaved seawater supplemented with filter-sterilized vitamins and inorganic nutrients. Cultures were incubated at 18 °C under constant light (mean intensity 100 μιτιοΙ m"2 s"1). Sterility was monitored by occasional inoculation into peptone-enriched media to check for bacterial growth. Diatom cells were harvested by centrifugation for approximately 15 min at 4000g, and pellets were rapidly frozen in liquid N2 and stored at -80 °C before further workup in the laboratory. The material (40 g) was extracted three times with MeOH to give a polar crude organic extract (420 mg). A portion of this extract (15 mg) was tested for antimicrobial or apoptosis induction. The crude organic extract was found active and subjected to fractionation using a solid-phase extraction cartridge (normal-phase silica) to give four fractions, F1 to F4, using hexane, CH2CI2, EtOAc, and MeOH as an increasingly hydrophilic solvent system series. The fraction eluting with EtOAc (F3) had antimicrobial activities. This fraction was further chromatographed on analytical RP HPLC (Phenomenex luna C18, 250 * 4.60 mm) using isocratic elution with 100% MeOH (flow rate 1 mL/min) to yield several pure fatty acids (10 mg average weight each), readily used for sterile compositions.
Example 2: Lipid Extraction
"Extraction and analysis were performed using the method of Zuniga et al. (2012) of 'direct transesterification with convectional heat'. Briefly, 14 % boron trifluoride in methanol and hexane was added to freeze-dried algal powder, and the mixture was incubated at 90 °C for 1 h (Zuniga et al. 2012). Gas chromatography (Varian GC 3900) was used to quantify the fatty acid methyl esters (Zuniga et al. 2012). The gas chromatograph used hydrogen gas as the carrier, and flame ionization was used as a detector. Retention times were compared to standards in order to identify peaks (Metherel et al. 2013)." (Ruffell et al., 2015)
Example 3: Siliceous frustules cell wall isolation and purification
The residual solid from extraction was recuperated. This white greenish solid contains the siliceous frustules cell wall. This residue was washed several times with distilled water. To eliminate organic substances from this residue solid, calcination method was chosen.
The purification of the cell wall can be done by heating in an oven. Siliceous substances and inorganic substances will not be calcinated but fatty acids and other organic substances will be calcinated and eliminated around 350-500 degree C, or preferably at 600 degree C. The white and grayish substance remaining after calcination was readily used for sterile compositions.
The medium composition can be adjusted during the culture of Phaeodactylum tricornutum and add titanium salt or titanium hydroxide on the media. The organism will incorporate titanium hydroxide or titanium oxide into the structure of frustules cell wall, and the purification of the cell wall obtained by this method can also be done with the same process described above.
Example 4: Bioassay
"All the bacterial strains were sub-cultured three times as a purity check before beginning each experiment. This procedure was done by transferring one healthy colony to a new plate of media using streak plating. Once colonies were established, this was repeated two more times before the culture was used. Cultures were incubated in the dark in two CanTrol Environmental Systems Limited Model CES-37 (Canada) on plates of trypticase soy agar medium (TSA;
Sigma-Aldrich) at 37 °C (for E. coli and S. aureus). A colony was transferred to liquid medium, tryptic soy broth (TSB), and incubated for 24 h in order to be used for spread plating. Fatty acids used in this experiment were purchased from NuChek Prep Inc. (USA) and had purity greater than 99 %, with the exception of arachidonic acid which was greater than 90 % purity. Fatty acid stock solutions of 100 mg mL-1 were prepared by dilution with 100 % ethanol. Certain fatty acids [10:0, 14:0, 16:0, 17:0, 18:0, 20:0, 22:1 (n-9), 23:0, 24:0, and 24:1 (n-9)] were heated to 50 °C to improve solubility in alcohol. Seven-millimeter-diameter paper discs [Becton, Dickinson and Company (Canada)] were loaded with appropriate volumes of stock solution to result in a disc containing 2000, 800, 250, or 25 ig of fatty acid. Tetracycline was used as a positive control and as a basis for comparison to determine how effective the fatty acids are relative to a typical dose of a common antibiotic (Heman-Ackah 1976). A tetracycline stock solution of 50 mg mL"1 was diluted with sterile water and used to prepare 5- and 30^g discs of tetracycline, the latter of which is the Clinical and Laboratory Standards Institute (CLSI) standard (Clinical and Laboratory Standards Institute 2006). Ethanol-soaked discs were additionally prepared as solvent controls. Disk diffusion was selected as the test method to be consistent with standard methodology used by the CLSI for evaluating antibiotic effectiveness against bacteria (Sader et al. 2007). Spread plating was used to disperse the bacteria on plates. Loaded discs and bacterial plates were placed in a biosafety cabinet at room temperature until dry; then, loaded discs were added to the bacterial plates. Bacteria were incubated in the dark on plates of TSA media for 48 h at 37 °C (for E. coli and S. aureus). Inhibition was quantified by measuring the zone of clearing around the discs to the nearest millimeter. The zone of inhibition was measured at 24 and 48 h during incubation (Dawson et al. 2002 and Naviner et al. 1999)." (Ruffell et al., 2015) Example 5: Scale up culture.
In order to get sufficient material for sterile compositions a scale up culture is needed. A novel method of cultivating algae was used which combines the positive features of open pond systems with those of enclosed photobioreactor algae production systems. The system produces a continuous supply of ultra-pure algae biomass in high volumes. The design enables full control of all cultivation parameters allowing to achieve optimum growing conditions for any algae species. As well as, a unique C02 delivery system is used that enhances delivery efficiency and minimizes C02 losses (GHG emissions) from the system.
As an example the following strains were cultured from the system: Chlorella, Scenedesmus.
Algal oil was produced and the majority of the oils are fatty acids that were readily used for sterile compositions.
Example 6: Sterilization method using fatty acid isolated from cultured algae
Phaeodactylum tricornutum
40g of fatty acid isolated from cultured algae Phaeodactylum tricornutum produced as described in examples 1 and 5 were mixed with 900g squeezed fresh orange. These products were mixed intensively in a mixer for 5min. The mixture were left in open atmosphere at room temperature 25 °C for 1 day and then tested for sterility from common bacteria as described in example 4. The products were found to be free or at least 5-log reduction from the following bacteria: E. coli, and S. aureus.
Example 7: Sterilization method using fatty acid isolated from cultured algae
Phaeodactylum tricornutum and Ti02 in the form of anatase or nanocrystalline
90g of fatty acid ranging from C12 to C22 isolated from cultured algae Phaeodactylum tricornutum and produced as described in example 1 and 5 were mixed with 900g squeezed fresh orange. These products were mixed intensively in a mixer for 5min. The mixture was added with 10g of Titanium Oxide Nanoparticles (Ti02, anatase, 99%, 15 nm) purchased from NanoAmor Europe. The mixture were left in open atmosphere at room temperature 25 °C for 1 day and then tested for sterility from common bacteria as described in example 4. The products were found to be free or at least 5-log reduction from the following bacteria: E. coli, S. aureus and specifically Agrobacterium tumefaciens.
Example 8: Sterilization method using fatty acid isolated from cultured algae
Phaeodactylum tricornutum and cuprous chloride.
99mg of fatty acid ranging from C12 to C22 isolated from cultured algae Phaeodactylum tricornutum and produced as described in example 1 and 5 were mixed with 900mg squeezed fresh orange. These products were mixed intensively in a mixer for 5min. The mixture was added with 0.2mg of cuprous chloride CuCI. The mixture were left in open atmosphere at room temperature 25 °C for 1 day and then tested for sterility from common bacteria as described in example 4. The products were found to be free or at least 5-log reduction from the following bacteria: E. coli, S. aureus and specifically Agrobacterium tumefaciens.
Example 9: Sterilization method using fatty acid isolated from cultured algae
Phaeodactylum tricornutum and Copper (Cu) Nanopowder APS: 70 nm.
90mg of fatty acid ranging from C12 to C22 isolated from cultured algae Phaeodactylum tricornutum and produced as described in example 1 and 5 were mixed with 900mg squeezed fresh orange. These products were mixed intensively in a mixer for 5min. The mixture was added with 0.1 mg of Copper (Cu) Nanopowder APS (70 nm) purchased from US Research Nanomaterials, Inc . The mixture were left in open atmosphere at room temperature 25 °C for 1 day and then tested for sterility from common bacteria as described in example 4. The products were found to be free or at least 5-log reduction from the following bacteria: E. coli, S. aureus and specifically Agrobacterium tumefaciens.
Example 10: Sterilization method using cultured algae Phaeodactylum tricornutum
100g of Cultured algae Phaeodactylum tricornutum produced as described in example 5 were ground with final particles size of 0.1 μηη. These greenish products were mixed intensively with 900g of corn flour. The mixture products were left in open atmosphere at room temperature 25 °C for 1 day and then tested for sterility from common bacteria as described in example 4. The products were found to be free or at least 5-log reduction from the following bacteria: E. coli, S. aureus.
Example 11 : Sterilization method using cultured algae Phaeodactylum tricornutum and Ti02 in the form of anatase or nanocrystalline
90g of Cultured algae Phaeodactylum tricornutum produced as described in example 5 were ground with final particles size of 0.1 m. These greenish products were mixed intensively with 900g of corn flour. The mixture was added with 10g of Titanium Oxide Nanoparticles (Ti02, anatase, 99%, 15 nm) purchased from NanoAmor Europe. The mixture products were left in open atmosphere at room temperature 25 °C for 1 day and then tested for sterility from common bacteria as described in example 4. The products were found to be free or at least 5-log reduction from the following bacteria: E. coli, S. aureus and specifically Agrobacterium tumefaciens.
Example 12: Sterilization method using cultured algae Phaeodactylum tricornutum and Copper (Cu) Nanopowder APS: 70 nm.
99g of Cultured algae Phaeodactylum tricornutum produced as discribed in example 5 were ground with final particles size of 0.1 m. These greenish products were mixed intensively with 900g of corn flour. The mixture was added with 0mg of Copper (Cu) Nanopowder APS (70 nm) purchased from US Research Nanomaterials, Inc. The mixture products were left in open atmosphere at room temperature 25 °C for 1 day and then tested for sterility from common bacteria as described in example 4. The products were found to be free or at least 5-log reduction from the following bacteria: E. coli, S. aureus and specifically Agrobacterium tumefaciens. Example 13: Sterilization method using cultured algae Phaeodactylum tricornutum and engineered Siliceous frustules cell wall
90g of Cultured algae Phaeodactylum tricornutum produced as described in example 5 were ground with final particles size of 0.1 μπι. These greenish products were mixed intensively with 900g of corn flour. The mixture was added with 10g of engineered Siliceous frustules cell wall as described in example 3. The mixture products were left in open atmosphere at room temperature 25 °C for 1 day and then tested for sterility from common bacteria as described in example 4. The products were found to be free or at least 5-log reduction from the following bacteria: E. coli, S. aureus and specifically Agrobacterium tumefaciens.
Example 14: Sterilization method using extract of cultured algae Phaeodactylum tricornutum and engineered Siliceous frustules cell wall
90g of extract of cultured algae Phaeodactylum tricornutum produced as described in examples 1 and 5 were mixed with 900g squeezed fresh orange. These products were mixed intensively in a mixer for 5min. The mixture was added with 10g of engineered Siliceous frustules cell wall as described in example 3. The mixture products were left in open atmosphere at room temperature 25 °C for 1 day and then tested for sterility from common bacteria as described in example 4. The products were found to be free or at least 5-log reduction from the following bacteria: E. coli, S. aureus and specifically Agrobacterium tumefaciens.
Example 15: Sterilization method using extract of cultured algae Phaeodactylum tricornutum and Ti02 in the form of anatase or nanocrystalline
90g of extract of cultured algae Phaeodactylum tricornutum produced as described in examples 1 and 5 were mixed with 900g squeezed fresh orange. These products were mixed intensively in a mixer for 5min. The mixture was added with 10g of Titanium Oxide Nanoparticles (Ti02, anatase, 99%, 15 nm) purchased from NanoAmor Europe. The mixture products were left in open atmosphere at room temperature 25 °C for 1 day and then tested for sterility from common bacteria as described in example 4. The products were found to be free or at least 5-log reduction from the following bacteria: E. coli, S. aureus and specifically Agrobacterium tumefaciens.
Example 16: Sterilization method using fatty acid isolated from cultured algae
Phaeodactylum tricornutum and engineered Siliceous frustules cell wall
90g of fatty acid ranging from C12 to C22 isolated from cultured algae Phaeodactylum tricornutum and produced as described in example 1 and 5 were mixed with 900g squeezed fresh orange. These products were mixed intensively in a mixer for 5min. The mixture was added with 10g of engineered siliceous frustules cell wall as described in example 3. The mixture products were left in open atmosphere at room temperature 25 °C for 1 day and then tested for sterility from common bacteria as described in example 4. The products were found to be free or at least 5-log reduction from the following bacteria: E. coli, S. aureus and specifically Agrobacterium tumefaciens.
Example 17: Data reported under pH> 6 for sterilization method using fatty acid isolated from cultured algae Phaeodactylum tricornutum and Ti02 in the form of anatase or nanocrystalline
It is known that acidic condition with pH≤ 5 may inhibit the growth of bacteria. The example 7 was repeated but with pH > 6 by addition of NaOH. 90g of fatty acid ranging from C12 to C22 isolated from cultured algae Phaeodactylum tricornutum and produced as described in example 1 and 5 were mixed with 900g squeezed fresh orange. These products were mixed intensively in a mixer for 5mn. The mixture was added with 10g of Titanium Oxide Nanoparticles (Ti02, anatase, 99%, 15 nm) purchased from NanoAmor Europe. The mixture were left in open atmosphere at room temperature 25 °C for 1 day and then tested for sterility from common bacteria as described in example 4. The products were found to be free or at least 5-log reduction from the following bacteria: E. coli, S. aureus and specifically Agrobacterium tumefaciens. Example 18: Sterilization method using fatty acid isolated from cultured algae Phaeodactylum tricornutum and Nitrogen and sulfur co-doped Ti02 it is known that the overall efficiency of Ti02 under natural sunlight is limited to the UV-driven activity (λ < 400 nm), shifting the absorption spectrum of Ti02 towards the visible-light range is possible by incorporation of no-metallic dopants into its lattice. For this purpose example 7 was repeated with a synthetic Nitrogen and sulfur co-doped Ti02. 90g of fatty acid ranging from C12 to C22 isolated from cultured algae Phaeodactylum tricornutum and produced as described in example 1 and 5 were mixed with 900g squeezed fresh orange. These products were mixed intensively in a mixer for 5min. The mixture was added with 10g of Nitrogen and sulfur co-doped Ti02 synthesized from Titanium Oxide Nanoparticles and using manual grinding with thiourea and annealing at 400 °C (J.A. Rengifo-Herrera, C. Pulgarin / Solar Energy 84 (2010) 37-43). The mixture were left in open atmosphere at room temperature 25 °C for 1 day and then tested for sterility from common bacteria as described in example 4. The products were found to be free or at least 5-log reduction from the following bacteria: E. coli, S. aureus and specifically Agrobacterium tumefaciens.
Example 19 : Use of Polenta sample
The use of polenta made by boiling cornmeal was also experimented. The purpose of this example is to verify different kind of food sample. Example 7 was repeated using polenta made by boiling cornmeal instead of squeezed fresh orange. 90g of fatty acid ranging from C12 to C22 isolated from cultured algae Phaeodactylum tricornutum and produced as described in example 1 and 5 were mixed with 900g polenta. These products were mixed intensively in a mixer for 5min. The mixture was added with 10g of Titanium Oxide Nanoparticles (Ti02, anatase, 99%, 15 nm) purchased from NanoAmor Europe. The mixture were left in open atmosphere at room temperature 25 °C for 1 day and then tested for sterility from common bacteria as described in example 4. The products were found to be free or at least 5-log reduction from the following bacteria: E. coli, S. aureus and specifically Agrobacterium tumefaciens. Example 20: Sterilization method using cultured algae Phaeodactylum tricornutum and Ti02 in the form of anatase or nanocrystalline and polenta as food sample
The following variant of example was also experimented. 90g of cultured algae Phaeodactylum tricornutum produced as described in example 5 were ground with final particles size of 0.1 pm. These greenish products were mixed intensively with 900g of polenta made by boiling cornmeal. The yellow greenish mixture was added with 10g of Titanium Oxide Nanoparticles (Ti02, anatase, 99%, 15 nm) purchased from NanoAmor Europe. The mixture products were left in open atmosphere at room temperature 25 °C for 1 day and then tested for sterility from common bacteria as described in example 4. The products were found to be free or at least 5-log reduction from the following bacteria: E. coli, S. aureus and specifically Agrobacterium tumefaciens.
Example 21 : Time dependent growth of bacteria
It is known that bacteria can grow over time. The result may present false reading on day one. The examples 7 and 1 1 were repeated under the same condition and sterilization compositions but the reading of the experiments were performed after 4 days instead of 1 day. The products were found to be free or at least 5-log reduction from the following bacteria: E. coli, S. aureus and specifically Agrobacterium tumefaciens.
Example 22: Combination of copper and Ti02 as disinfectants
It is known that disinfectants may act efficiently when combined. The following variant was exemplified by using combination of Copper (Cu) Nanopowder APS (70 nm) and Titanium Oxide Nanoparticles (Ti02, anatase, 99%, 15 nm).
90g of fatty acid ranging from C12 to C22 isolated from cultured algae Phaeodactylum tricornutum and produced as described in example 1 and 5 were mixed with 900g squeezed fresh orange. These products were mixed intensively in a mixer for 5min. The mixture was added with 5g of Titanium Oxide Nanoparticles (Ti02, anatase, 99%, 15 nm) purchased from NanoAmor Europe and 0.05mg of Copper (Cu) Nanopowder APS (70 nm) purchased from US Research Nanomaterials, Inc.. The mixture were left in open atmosphere at room temperature 25 °C for 1 day and then tested for sterility from common bacteria as described in example 4. The products were found to be free or at least 5-log reduction from the following bacteria: E. coli, S. aureus and specifically Agrobacterium tumefaciens.
It was found that the combination of Copper and Ti02 is 2 times potent than Ti02 alone and 2 times potent than Copper alone.
Useful ranges
It is proven from the above examples that several variants of the method and compositions are possible. The main components are fatty acids, extract of algae or cultured algae and the disinfectants are Ti02, copper in the form of nanoparticles or engineered Siliceous frustules cell wall. The weight percentage ranges of all components depend on the form of the material to be sterilized.
The following useful ranges are established, weight percentage: 90-95 % of human or animal food, 4-10% of fatty acid, extract of algae or cultured algae and 0-1 % of disinfectants selected from Ti02, copper in the form of nanoparticles or engineered Siliceous frustules cell wall.
Examples
4.2% of fatty acids , 95.7% of squeezed fresh orange
9% of fatty acids , 90% of squeezed fresh orange and 1 % of Ti02 nanoparticles
9.9% of fatty acids, 90% of squeezed fresh orange and 0.02% of cuprous chloride
9.1 % of fatty acids, 90.8% of squeezed fresh orange and 0.01 % of copper in the form of nanoparticles
10% of cultured algae, 90% of corn flour 9% of cultured algae, 90% of corn flour and 1% of Ti02 nanoparticles
9.9% of cultured algae, 90.08% of corn flour and 0.001 % of copper in the form of nanoparticles
9% of cultured algae, 90% of corn flour and 1 % of engineered siliceous frustules cell wall
9% of extract of algae, 90% of squeezed fresh orange and 1% of engineered Siliceous frustules cell wall
9% of extract of algae, 90% of squeezed fresh orange and 1% of Ti02 nanoparticles
9% of fatty acids, 90% of squeezed fresh orange and 1% of engineered Siliceous frustules cell wall
9% of fatty acids, 90% of squeezed fresh orange and 1 % of Nitrogen and sulfur co-doped Ti02 nanoparticles
9% of fatty acids, 90% of polenta and 1 % of Ti02 nanoparticles 9% of cultured algae, 90% of polenta and 1 % of Ti02nanoparticles
9% of fatty acids, 90.45% of squeezed fresh orange , 0.5% of Ti02 nanoparticles and 0.005 % of copper in the form of nanoparticles
Whereas specific embodiments are herein shown and described, variations are possible.
Accordingly, the invention should be understood to be limited only by the claims, purposively construed.

Claims

A method of sterilization and product preparation, the method comprising the step of mixing the product with (i) algae or with an algae derivative; and (ii) a disinfectant adapted for Agrobacterium tumefaciens.
A method according to claim 1 , wherein the product is one of grits, polenta, corn meal, corn flour and orange juice.
A method according to claim 1 , wherein the algae is Phaeodactylum tricornutum.
A method according to claim 1 , wherein the algae is one of Rhodella maculata (Rhodophyta), Boekelovia hooglandii (Heterokontophyta), Goniochloris sculpta (Heterokontophyta), Chlorideila simplex (Heterokontophyta), Ascophyllum sp., Fucus sp., Himanlhalia sp., Undaria sp., and Laminaria sp.
A method according to claim 1 , wherein the disinfectant is one of
Ti02
copper
copper-containing sulfur doped Ti02 ; and
copper, silver and nickel-enriched Ti02
A method according to claim 1 , wherein the algae derivative is an extract from algae containing fatty acid, the fatty acid ranging from C12 to C22.
A method according to claim 1 , wherein the algae derivative is an extract from algae having added with purified siliceous frustules cell wall.
A method according to claim 1 , wherein the algae derivative is an extract added with purified and engineered siliceous frustules cell wall to contain Ti(OH)4 in situ.
9. A method according to claim 1 , wherein said disinfectant is Ti02 in the form of anatase or nanocrystalline.
10. A method according to claim 1 , wherein the resultant mixture has
90-95 wt % product.
11. A method according to claim 10, wherein the resultant mixture has 4-10 wt% algae.
12. A method according to claim 1 1 , wherein the resultant mixture has up to 1 % disinfectant.
13. A method according to claim 12, wherein extract is an omega 4 polyunsaturated fatty acid isolated from Phaeodactylum tricornutum.
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