GB2516702A - Colorimetric Phytic Acid Assay - Google Patents
Colorimetric Phytic Acid Assay Download PDFInfo
- Publication number
- GB2516702A GB2516702A GB1317332.3A GB201317332A GB2516702A GB 2516702 A GB2516702 A GB 2516702A GB 201317332 A GB201317332 A GB 201317332A GB 2516702 A GB2516702 A GB 2516702A
- Authority
- GB
- United Kingdom
- Prior art keywords
- phytic acid
- composition
- solution
- precipitate
- range
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- IMQLKJBTEOYOSI-GPIVLXJGSA-N Inositol-hexakisphosphate Chemical compound OP(O)(=O)O[C@H]1[C@H](OP(O)(O)=O)[C@@H](OP(O)(O)=O)[C@H](OP(O)(O)=O)[C@H](OP(O)(O)=O)[C@@H]1OP(O)(O)=O IMQLKJBTEOYOSI-GPIVLXJGSA-N 0.000 title claims abstract description 122
- 235000002949 phytic acid Nutrition 0.000 title claims abstract description 122
- IMQLKJBTEOYOSI-UHFFFAOYSA-N Phytic acid Natural products OP(O)(=O)OC1C(OP(O)(O)=O)C(OP(O)(O)=O)C(OP(O)(O)=O)C(OP(O)(O)=O)C1OP(O)(O)=O IMQLKJBTEOYOSI-UHFFFAOYSA-N 0.000 title claims abstract description 99
- 239000000467 phytic acid Substances 0.000 title claims abstract description 98
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- INEWUCPYEUEQTN-UHFFFAOYSA-N 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid Chemical compound OS(=O)(=O)CC(O)CNC1CCCCC1 INEWUCPYEUEQTN-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/66—Phosphorus compounds
- A61K31/661—Phosphorus acids or esters thereof not having P—C bonds, e.g. fosfosal, dichlorvos, malathion or mevinphos
- A61K31/6615—Compounds having two or more esterified phosphorus acid groups, e.g. inositol triphosphate, phytic acid
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/06—Phosphorus compounds without P—C bonds
- C07F9/08—Esters of oxyacids of phosphorus
- C07F9/09—Esters of phosphoric acids
- C07F9/117—Esters of phosphoric acids with cycloaliphatic alcohols
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/0098—Plants or trees
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/02—Food
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/18—Water
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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Abstract
A method for detecting an amount of phytic acid in a composition comprises: (a) providing a composition having phytic acid, (b) combining the composition of (a) with an iron solution; (c) separating out a precipitate from (b); (d) adding a wash solution to the precipitate of (c); (e) separating out a second precipitate from (d); (f) adding a sulphosalicylic acid and a buffer solution at a pH from 6 to 12 to the second precipitate of (e); (g) detecting an absorbance of the solution of (f) and comparing the solution to a standard phytic acid sample. The composition having phytic acid maybe an animal feed or supplement, food or dietary supplement for human consumption, urine, soil sample, waste sample, grain, cereal, soybeans, plants, dough, or a sample from an industrial process. Preferably, the absorbance is measured using a visual inspection, spectrophotometer, or a colour guide.
Description
COLORIMETRIC PHYTIC ACID ASSAY
TECHNICAL FIELD
This invention is directed to methods of chemical testing or analysis. An embodiment of the invention relates to phytic acid assays. An embodiment of this invention is to provide a colorimetric assay for the quantification of phytic acid. In another embodiment the phytic acid assay can be used in the field as a testing kit. In another embodiment the testing kit comprises a color guide for identifying the amount of phytic acid in the composition being tested. In another embodiment the phytic acid content is used to determine a phytase dosage. In another embodiment, the phytase is added to an animal feed or supplement, food or dietary supplement for human consumption, a urine sample, soil sample. waste sample, grain, cereal, soybeans, plants, dough, a liquid sample, an oil samp'e, a fermentation sample, or an industrial process such as ethanol production.
Feed stocks, which are the raw ingredients of the animal feed, come from a variety of sources including: plants; animals; edible by-products; and additives, such as vitamins, minerals, enzymes, and other nutrients (SAPKOTA; Environ Health Perspect. 2007 May; 115(5): 663-670).
Phytic acid, also known as phytate, myo-inositol (1,2,3,4,5,6) hexakisphosphate, or 1P6; is known to be present in plant seeds,roots tubers, and in animal feed products having plant parts. Plant based food and feed products such as: wheat, barley, triticaie, rye, corn, sorghum soybean meal, and canola meal all contain phytic acid. Phytic acid is known to bind or chelate ions such as iron, calcium, zinc, potassium, and/or magnesium. Chelating of phytate and metal ions is known to decrease the nutritional value of the food or feed. Phytase is often added to the food or feed in order break down the phytate and improve digestibility of beneficial nutrients that improve the health and growth of animals.
Animal feed additives, such as enzymes, are designed to increase the nutritional value of the feed by releasing nutrients and allowing increased absorption of essential vitamins and minerals in the animal, which in turn, enhances animal product yield, while reducing harmful materials in animal waste.
Enzymes used as additives in the manufacturing of food and feed include, but are not limited to: a phytase, cellulase. lactase, lipase, protease, catalase, xylanase, beta-glucanase, mannanase, amylase. amidase, epoxide hydrolase, esterase, phospholipase, transaminase, amine oxidase, cellobiohydrolase, ammonia lyase, or any combination thereof.
A variety of animals can benefit from animal feed containing an enzyme including: non-ruminant animals. e.g. poultiy. broilers, birds, chickens, layers. turkeys. ducks, geese. and fowl; ruminant animals e.g. cows, cattle, horses, and sheep; pigs, swine, piglets, growing pigs, and sows; companion animals including but not limited to: cats, dogs, rodents, and rabbits; fish including but not limited to salmon, trout, tilapia, catfish and carp; and crustaceans induding but not limited to shrimp and prawn.
Knowledge of the substrate phytate) and reaction conditions is essential for proper dosage of enzymes, such as phytase, for animal feed. See, Classen "Ingredient Considerations, total phytate concentrations and susceptibility of phytate to hydrolysis," (2010) Proceedings of the 1st International Phytase Summit. A number of quafitative and quantitative analytical methods are known for determining an amount of phytate in an animal feed composition.
However, no single analytical method will fulfill the needs for all circumstances. See. Oberleas, "Strengths and weaknesses of current phytate analytical methods" (2010) Proceedings of the 1st International Phytase Summit. Oberleas further reports that interest in qualitative methods has declined with advancements in technology. In addition, Oberleas reports that quantification methods, such as HPLC. are expensive to acquire and maintain and are not accessible to many laboratories, especially in developing countries.
Therefore, a need exists in the art to provide a phytic acid assay that can quickly, accurately, and consistently measure phytic acid levels of a composition containing phytic acid such as an animal feed. An embodiment of this invention is to provide an assay for the quantification of phytic acid at high pM in a solution. In another embodiment the phytic acid assay can be used in the field as a testing kit. hi another embodiment the testing kit comprises a color guide for identifying the amount of phytic acid in the composition being tested. In another embodiment phytic acid is measured in a composition such as an animal feed or supplement, food or dietary supplement for human consumption a baking product (dough), urine, waste, soybeans, or any composition comprising phytate.
In addition, it is known that measuring phytic acid is important in the baking industry.
See, Frontela "Phytic acid content and In vitro" iron, calcium and zinc bioavailability in bakery products: The effect of processing" Journal of Cereal Science, Volume 54, Issue 1. July 2011, Pages 173-179. In one embodiment, colorirnetric phytic acid assay can be used to test the compositions in a baking process.
A procedure for determining the amount of urinaly phytate in the prevention of calcium renal stone formation was described in Costa-Bauza, A., et. al. "A simple and rapid colorimetric method for deteimination of phytate in urine" (2012) Urological Research, 40 (6), pp. 663-669.
In another embodiment, the colorimetric phytic acid assay can be used to test a urine sample.
Methods of detecting phytic acid content for breeding and genetic studies of soybeans was determined by comparison study of different methods including: a modified colorimetric (Wade Reagent) method, an Anion Exchange Colunm Method AEC); a High-performance Liquid Chromatography Analysis (HPLC); and Nuclear Magnetic Resonance Analysis (NMR).
Gao, et. al., "A modified Colorimetric Method for Phytic Acid Analysis in Soybean" (2007) Crop Science Vol. 47, pages 1797-1803. In another embodiment, the colorimetric phytic acid assay can be used to test a plant sample, such as a wheat, barley. triticale, lye. corn, sorghum soybean meal, and canola meal.
Industrial processes. such as ethanol production or oilseed processing, are known to use a variety of feedstocks having phytic acid such as grains, ceflulosic material, or oilseeds. Phytic acid is known to create sludge know as scale or fouling that can damage industnal production equipment or reduce performance of the facility. In one embodiment the colorimetric phytic acid assay can be used to test a sample from the industry production facility and used to determine the amount of phytase that needs to be added to the process in order to remove the phytic acid. In another embodiment, the sample can be from any step in the ethanol production process including but not limited to: storage, milling, slurry tanks, liquefaction, fermentation, distillation, dehydration or evaporation, holding tanks, separation, thin stillage, or co-products. Ethanol known to be used in a variety of products such as: fuel ethanol, beverages, distilled spirits, alcohol, personal care products, pharmaceutical industry, cleaning products, or food products such as extracts, and flavorings. Co-products from ethanol production and oilseed processing include distillers grain such as dried distillers grain (DDG), wet distillers grain (WDG), dried distillers grain with solubles (DDGS). Distillers grain is used for high quality feedstuff for animals such as cattle, swine, poultry, and aquaculture. Distillers grain is known to contain phytic acid, which as mentioned above, is known to decrease the nutritional value of the feed.
Thus the colorimetric phytic acid assay can be used to test distillers grains and used to determine the amount of phytase that needs to be added in improve the nutritional value of the feed.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING
Figure 1: Shows a comparison of phytic acid content from sample A. which was an HCI extraction and Sample B, which was a water extraction. The samples were analyzed for Phytic Acid content using three different methods: ICP, HPLC, and the SSA colorimetric phytic acid method of this invention (the colorimetric suspensions were also analyzed using ICP).
Figure 2: Shows an ICP measurement comparison of Phytic Acid standards put through the Sulfosalicylic Acid method of the invention (PA std in SSA fron/Phytate Pellet and re-suspended) and PA std in iNHCL without Sulfosalicylic Acid. The figure shows the same amount of phosphorus is measured in the colorimetric re-suspension samples from the Sulfosalicylic Acid method of the invention as in the phytic acid standard from which the colorimetric samples were derived.
Figure 3: Shows a color guide for the visual inspection for a yellow color having low amounts of phytate in the range from about 0.1mM to about 0.75mM; an orange color for modest amounts phytic acid in the range from about 1.0mM to about 5.0mM or a red color for large amounts phytic acid in the range from about 6.0mM to about 8.0mM.
DETAILED DESCRIPTION OF THE INVENTION
An "enzyme" as used herein refers to at least any enzyme that can be used as an additive for animal feed or supplement or food or dietary supplement for human consumption. For example, enzymes useful in the present invention include, but are not linñted to: a phytase, lactase, Upase. protease, catalase, xylanase, cellulase, glucanase, mannanase, amylase. amidase, epoxide hydrolase, esterase, phospholipase, transaminase, amine oxidase, cellobiohydrolase, ammonia lyase, or any combination thereof.
A "phytase" is an enzyme that catalyzes the removal of one or more phosphate groups from a phytate substrate. In another embodiment, a phytase is a phosphoric monoester hydrolase enzyme that catalyzes hydrolysis of phytic acid (myo-inositol-hexakisphosphate) to phosphate and myo-inositol having fewer than six phosphate groups. According to the recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB) and Bairoch A., "The ENZYME database in 2000," Nucleic Acids Res 28:304- 305(2000), a phytase may be described in a variety of names and identifying numbers. In another embodiment, a phytase is characterized as having Enzyme Commission (EC) number EC 3.1.3.8, and are also referred to as: 1-phytase; myo-inositol-hexakisphosphate 3-phosphohydrolase; phytate 1-phosphatase; phytate 3-phosphatase; orphytate 6-phosphatase. In another embodiment, a phytase is characterized as EC 3.1.3.26, also referred to as: 4-phytase; 6-phytase (name based on IL-numbering system and not ID-numbering); or phytate 6-phosphatase. In another embodiment, a phytase is characterized as PC 3 3.72, also referred to as 5-phytase. In another embodiment, a phytase is a histidine acid phosphatase (HAP); a p-propeller phytase; purple acid phosphatase (PAP); and protein tyrosine phosphatase (PTPs). In some embodiments, a phytase is described using nomenclature know in the art.
In one embodiment the invention is a colorimetric assay for the quantification of phytic acid which can be used in the field as a testing kit with a color guide.
The industry standard methods for phytic acid detection (animal feed, cereals, etc.) are indirect methods based on measuring the excess iron remaining from the covalent binding of iron to phytic acid. The indirect methods add a known amount of iron to phytic acid; the phytic acid will bind a portion of the iron; and the remaining unbound or excess iron is measured. The amount of iron bound by phytic acid can be then be calculated by subtracting the remaining unbound or excess iron from the known amount. However, in an embodiment of this invention the indirect methods are simplified by precipitating and then re-solubilizing phytic acid (and the iron bound to the phytic acid) and directly measuring the amount of iron bound to the phytic acid, rather than measuring the amount of excess iron remaining after the reaction. In another embodiment the phytic acid iron complex is in a solution and the color of the solution colTelates to the concentration of phytic acid in s&ution. In another embodiment the concentration is used to determine a dosage of phytase that should be added to a composition comprising phytic acid.
Vanous methods are known from literature that discloses how phytic acid phytate) can be separated from samples by binding it with iron and precipitating it out of solution. However, an embodiment of this invention uses Sulfosalicylic Acid (SSA) to solubilize the iron-phytate pellet into solution for analysis. In another embodiment, the instant assay uses Sulfosalicylic Acid (SSA) and Sodium hydroxide NaOH) to solubilize the iron-phytate pellet into solution for analysis. In another embodiment of this invention, the process can detect phytate levels from about 0.1mM to about 8.0mM.
Phytate is a chelator which binds minerals such as iron; therefore, it can be precipitated out of a solution with the addition of iron. Sulfosalicylic Acid (SSA) is also a chelator of minerals. Sulfosalicylic Acid will bind iron molecules at varying amounts based on the pH value. Methods known in the ar, use Sulfosalicylic Acid that will bind one molecule of iron from phytic acid at pH<4 for very small concentrations of phytic acid. However, an embodiment of this invention tests phytic acid for higher concentrations at a pH >4. lii one embodiment the p1-I is a high p1-I such as pI-I6, pH7. p1-I 8, pH9, pi-IIO, pHI I, or pH 12. At pH>4 Sulfosalicylic Acid will strongly bind two or three molecules of iron from phytic acid which allows the phytic acid to solubilize in a solution. Using.5M SSA with 20mM methylpiperazine pH 10 using Sodium Hydroxide (or an alternate buffer solution such as: Phosphoric acid (H3P04). tris-HC1 (trisaminornethane ((HOCH2)3CNH2)), bis-tris (2-[Bisamino]-2--I,3-propanediol), propane (C3H8). boric acid, ammonia, carbonate, glycine, CAPS, CAPSO. at or above: pH 8, pH 9, pHIO, pHI I, pHI2), the iron-phytate pellet will solubilize. Iii another embodiment, the amount of phytate present will be measured on a color development scale in shades of red (yellow for low amounts of phytate from about 0.1mM to about 0.75mM; orange for modest amounts of phytate from about 1.0mM to about 5.0mM; and red for high amounts of phytate from about 6.0mM to about 8.0mM). In another embodiment, the method for quantitating phytic acid (shade of red) would be detection within a range from about 485 nm to about 545 nm using a spectrophotometer (cuvette or p'ate based format).
In one embodiment the method for detecting an amount of phytic acid in a composition comprises: (a) providing a composition having phytic acid; (b) combining the composition of (a) with an iron solution; (c) separating out a precipitate from (b); (d) adding a wash solution to the precipitate of (c); (e) separating out a second precipitate from (d); (f) adding a sulfosalicylic acid and a buffer solution at a pH from 6 to 12 to the second precipitate of (e); (g) detecting an absorbance of the solution of (0 and comparing the solution to a standard phytic acid sample.
In one embodiment the method for detecting an amount of phytic acid in a composition comprises: (a) providing a composition having phytic acid; (b) combining the composition of (a) with an iron solution; (c) separating out a precipitate from (b); (d) adding a wash solution to the precipitate of (c); (e) separating out a second precipitate from (d); (0 adding a second wash solution to the second precipitate from (e); (g) separating out a third precipitate from (1); (h) adding a sulfosalicylic acid and a buffer solution at a pH from 6 to 12 to the third precipitate of (g); (Ig) detecting an absorbance of the solution of (h) and comparing the solution to a standard phytic acid sample.
In another embodiment, the method for detecting an amount of phytic acid in a composition can be done in a different order than the order listed above. For example, the composition having phytic acid can be combined with the iron solution in a single step. In another embodiment, the separation of solution and the precipitate can be with centrifuge, a filtration, or other methods known to those skilled in the art.
In one embodiment, the composition having phytic acid is an animal feed or supplement, food or dietary supplement for human consumption; urine, soil sample, waste sample, grain, cereal, soybeans. plants. dough, a liquid samp'e, a sample from an industrial process, a distillers grain, and/or any combination thereof. In another embodiment, the phytic acid is being measured in an ethanol processing facility. In another embodiment, the ethanol produced using grain or starch. In another embodiment, the ethanol is produced using lignocellulose material. In another embodiment, the grain, starch, or lignocellulosic material is a composition comprising phytic acid.
In one embodiment, the iron solution is a composition comprising: FeC13 (h-on (III) Chloride), H20 (water), and HC1 Hydrochloric acid). In another embodiment, the FeCl3 is within a range of from about 0.2% to about 1.0%. In another embodiment, the FeCI3 is 0.4%. In another embodiment, the iron s&ution comprises MCI within a range from about 0.167M 0.5 to about lOM. In another embodiment, the HCI is 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, i.OM, or anywhere within the range from about 0.lM to about 1GM.
In one embodiment, the wash solution I is a composition comprising: 1-ICI and Na2SO4 (sodium sulfate). In another embodiment, the MCI in wash solution I is within a range from about DiM to about 1.DM. In another embodiment, the HC1 in wash solution 1 is 0.167M. In another embodiment, the Na2SO4 in wash solution 1 is within a range from about 1.2% to about 10%. In another embodiment, the Na2SO4 in wash solution I is 4%.
In one embodiment, the wash solution 2 is a composition comprising: HCI in deionized water. In another embodiment, the HCI in wash solution 2 is within a range from about 0.1 to about lM. In another embodiment, the HCI in wash solution 2 is 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.SM, 0.9M, or 1DM.
In one embodiment, the sulfosalicylic acid solution is a composition comprising: sulfosalicylic acid (SSA) and 1-methylpiperazine. In another embodiment, the sulfosalicylic acid in the sulfosalicylic acid solution is within a range from about 0.IM to about l.OM. In another embodiment, the sulfosalicylic acid in the sulfosalicylic acid solution is 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, or 1.OM. In another embodiment, the 1-methylpiperazine in the sulfosalicylic acid solution is within a range from 0.O1M to 0.2M. In another embodiment, the 1-methylpiperazine is DiM, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, or 1DM. In another embodiment pH of the sulfosalicylic acid solution is within a range from about pH 6 to about pH 12. In another embodiment, the pH of the sulfosalicylic acid solution is pH 8, pH 9, pH 10, pH 11, or pH 12. In another embodiment, the pH of the sulfosalicylic acid solution is adjusted with NaOH, wherein the adjustment is within the range from about DIM to about 2.OM.
In one embodiment, the absorbance is measured using a visual inspection, a spectrophotometer, a color guide, or other methods known to those skilled in the art. In another embodiment, the visual inspection is yellow color for low amounts of phytate in the range from about 0.1mM to about 0.75mM. In another embodiment, visual inspection is an orange color for modest amounts phytic acid in the range from about 0.76mM to about 50mM. In another embodiment, visual inspection is red color for large amounts phytic acid in the range from about 5.1mM to about 8.0mM. In another embodiment, the color scale is on a card, or electronic device, wherein the color scale describes the concentration of phytic acid in solution. In another embodiment, the concentration of phytic acid is used to determine a dosage of phytase. In another embodiment, the phytase is added to an animal feed or supplement, food or dietary supplement for human consumption, a urine sample, soil sample, waste sample, grain, cereal, soybeans, plants, dough, a liquid sample, an oil sample, a fermentation sample, or an industrial process such as ethanol production
EXAMPLES
Reagents & Materials: I Normal Hydrochloric Acid (Fischer Chemical Cat.# UN1789); Sodium Sulfate (Sigma-Aldrich Cat.# S6547); 5-Sulfosalicylic Acid (Sigma-Aldrich Cat.# 390275); Iron III Chloride Hexahydrate (Sigma-Aldrich Cat.# 236489); Phytic Acid Dodecasodium Salt Hydrate (Sigma-Aldrich Cat.# P0109); 15 mL polypropylene conical tube (BD Falcon, 17 x 120mm style); 50 mL polypropylene conical tube (BD Falcon, 30 x 115 mm style); 1.7 mL Eppendorf tube (National Scientific Cat.#CNI700-BP); and NaOH Sigma-Aldrich Cat.# S5881). I -methylpiperazine (Sigma-Aldrich Cat.#l 30001) Standards & Solutions: Iron Solution: 0.4% FeC13 6H20 in 0.167% HC1; Wash Solution #1: 0.167M HC1 + 4% Na2SO4; Wash Solution #2: 0.SM HC1 in deionized H20; Sulfosalicylic Acid Solution: 0.SM Sulfosalicyhc Acid in 20mM 1-methylpiperazine pH to 0.5 with NaOFI; Phytic Acid Standard in lN HC1 at the following concentrations: 0.125mM, 0.25mM, 0.5mM, 1mM.
2mM. and 4mM.
Inductively coupled plasma emission spectroscopy (I CP) AOAC Official Method 985.01. in Official Methods of Analysis of AOAC International, 16th edition. Volume I Chapter 3, p. 4.
High-performance Liquid Chromatography (HPLC) Rounds: "Anion-Exchange high-performance liquid chromatography with post-column detection for the analysis of phytic acid and other inositol phosphates" Journal of Chromatography A, 653 (1993) p. 148-152.
Example 1: Extraction from Chicken Feed into Aqueous Solution and Sample Preparation Weigh Sg of a sample comprising phytic acid, such as an animal feed pellet into a 50 mL polypropylene conical tube. Add 20 mL of IN HC1. Shake the mixture at 30°C for 1 hour at 220 rpm. Centrifuge for 5 minutes at 4000 RPM to separate aqueous phase from the solid. Transfer aqueous phase into a clean 50 mL tube. In some embodiments. Na2SO4 can be added at 1.25% (1.0-5.0%). In another embodiment, TCA can be used as an alternative for extractions at 5% (I-10%) Example 2: Method of Detecting Phytic acid in a composition comprising phytic acid Add 0.5 mL of the supernatant from the sample preparation and 0.5 mL of Iron Solution to a 1.7 mL Eppendorf tube. Boil for 15 minutes. Ice for 15 minutes. Centrifuge for 2 minutes at 6000rpm. Pull off and discard supernatant.
Resuspend the iron-phytate pellet in I mL of Wash Solution #1 using pipette. Mix at 37°C for 10 minutes. In another embodiment. Wash solution #1 can be vortexed (mixed) and allowed to sit for 10mm; or it can be done at room temp. Centrifuge for 2 minutes at 6000rpm. Pull off and discard supematant.
Resuspend the iron-phytate pellet in 1 mL of Wash Solution #2 using pipette. Mix at 37°C for I hour. In another embodiment, Wash solution #2 can be vortexed (mixed) and allowed to sit for 10mm; or it can be done at room temp. Centrifuge for 2 minutes at 6000rpm. Pull off and discard supernatant.
Resuspend the iron-phytate pellet in I mL of Wash Solution #2 using pipette. Mix at 37°C for 10 minutes. In another embodiment, Wash solution #2 can be vortexed (mixed) and allowed to sit for 10mm; or it can be done at room temp. Centrifuge for 2 minutes at 6000rpm. Pull off and discard supernatant.
Resuspend iron-phytate pellet in 1 mL of Sulfosalicylic Acid solution using pipette, Mix at 37°C for 15 minutes, or vortex for I 0mm, or until there is no precipitate. Measure absorbance at 505nm of a 100 uL aliquot. Plot a standard curve using the Phytic Acid standard samples.
Determine chicken feed sample Phytic Acid concentration by plugging absorbance value into standard curve slope.
All numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth herein are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of any claims in any application claiming priority to the present application, each numencal parameter should be construed in light of the number of significant digits and ordinary rounding approaches.
The above description discloses several methods and materials of the present invention.
This invention is susceptible to modifications in the methods and materials, as well as alterations in the fabrication methods and equipment. Such modifications will become apparent to those skilled in the art from a consideration of this disclosure or practice of the invention disclosed herein. Consequently. it is not intended that this invention be limited to the specific embodiments disclosed herein, but that it cover all modifications and alternatives coming within the true scope and spirit of the invention.
All references cited herein. induding but not Urnited to published and unpublished applications, patents, and literature references, are incorporated herein by reference in their entirety and are hereby made a part of this specification. To the extent publications and patents or patent applications incorporated by reference contradict the disdosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.
Claims (14)
- CLAIMS1. A method for detecting an amount of phytic acid in a composition comprising: (a) providing a composition having phytic acid; (b) combining the composition of (a) with an iron solution; (c) separating out a precipitate from (b); (d) adding a wash solution to the precipitate of (c); (e) separating out a second precipitate from (d); (0 adding a sulfosalicylic acid and a buffer solution at a pH from 6 to 12 to the second precipitate of (e); Kg) detecting an absorbance of the solution of (f) and comparing the solution to a standard phytic acid sample.
- 2. The method of claim 1, wherein the composition having phytic acid is an animal feed or supplement, food or dietary supplement for human consumption; urine, soil sample, waste sample, grain, cereal, soybeans, plants, dough. a liquid sample, an industrial sample, or any combination thereof 3. The method of claim I, wherein the iron solution is a composition comprising: FeCI3, water.and HC1 4. The method of claim 3, wherein the FeC13 is within a range of from 0,2% to 1.0%; and the HClis within a range from 0.IM to 1.OM.5. The method of claim 1, wherein the wash solution is a composition comprising: HC1 and Na2SO4, deionized water, or a combination thereof.6. The method of claim 5, wherein the HCI is within a range from 0. 1M to 1.OM.7. The method of claimS, wherein the Na2SO4 is within a range from 1.2% to 10.0% 8. The method of claim 1, further comprises an additional wash step after step (e) followed by a separation of a third precipitate.9. The method of claim 1, wherein the buffer is a composition comprising: 1-methylpiperazine.10. The method of claim 9, the 1-methylpiperazine is within a range from 0.OlM to 0.2M at a pH from 6 to 12.11. The method of claim 1, wherein the sulfosalicyfic acid is within a range from 0.1 M to I.OM.12. The method of claim 1, wherein the absorbance is measured using a visual inspection, a spectrophotometer, or a card describing absorbance.13. The method of claim 12, wherein the visual inspection is yellow for low amounts of phytic acid from 0.1mM to 0.75mM; orange for modest amounts of phytic acid from 0.76mM to 5.0mM; or red for high amounts of phytic acid from 5.1mM to 8.0mM.14. The method of claim 1, further comprising calculating an amount of phytase to be added to the composition comprising phytic acid.CLAIMS1. A method for detecting an amount of phytic acid in a composition comprising: (a) providing a composition having phytie acid; (h) combining the composition of (a) with an iron solution: (c) separating out a prccipitate from (b): (d) adding a wash solution to the precipitate of (c); (e) separating out a second precipitate from (d); (0 adding a sulfosalicylic acid and a buffer solution at a pH from 6 to 12 to the second precipitate of (e); (g) detecting an absorbance of the solution of (f) and comparing the solution to a standard phytic acid sample.2. The method of claim 1, wherein the composition having phytic acid is an animal feed or supplement, food or dietary supplement for human consumption; urine, soil sample. waste sample, grain, cereal, soybeans. plants. dough, a liquid sample, a sample from an industrial process. or any combination thereof.
- 3. The method of claim I, wherein the iron solution is a composition comprising: FeCl3, water, Osj and HCI
- 4. The method of claim 3. wherein the FeC13 is within a range of from 0.2% to 1.0%; and the HC1 is within a range from 0.1M to 1.OM.
- 5. The method of claim 1, wherein the wash solution is a composition comprising: 1-ICI and Na2SO4. deionized water, or a combination thereof.
- 6. The method of claim 5, wherein the HCI is within a range from 0. 1M to 1.OM.
- 7. The method of claim 5. wherein the Na2SO4 is within a range from 1.2% to 10.0%
- 8. The method of claim 1, further comprises an additional wash step after step (e) followed by a separation of a third precipitate.
- 9. The method of claim 1, wherein the buffer is a composition comprising: I -methylpiperazine.
- 10. The nicthod of claim 9, the l-methylpipcrazinc is within a range froni 0.OlM to 0.2M at a pH from 6 to 12.
- 11. The method of claim I, wherein the sulfosalicyhc acid is within a range from 0.1 M to I.OM.
- 12. The method of claim 1, wherein the absorbance is measured using a visual inspection, a spcctrophotomctcr, or a card describing absorhance.
- 13. The method of claim 12, wherein the visual inspection is yellow for low amounts of phytic acid from 0.1mM to 0.75mM; orange for modest amounts of phytic acid from 0.76mM to 5.0mM; or red for high amounts of phytic acid from 5.1mM to 8.0mM.
- 14. The niethod of claim 1, further comprising calculating an amount of phytase to be added to the composition comprising phytic acid. (4
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| PCT/US2014/047290 WO2015010084A1 (en) | 2013-07-19 | 2014-07-18 | Colorimetric phytic acid assay |
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| US11499952B2 (en) | 2018-05-11 | 2022-11-15 | Premex, Inc. | Process for quantification of metal amino acid chelates in solutions and solids |
| CN109975237A (en) * | 2019-03-04 | 2019-07-05 | 浙江大学 | The quickly method of measurement rice milled rice flour phytic acid |
| CN112326848B (en) * | 2020-10-23 | 2022-11-29 | 杭州师范大学 | Methyldiazomethane methyl esterification phytic acid analysis method based on trimethylsilyl |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| SU1051415A1 (en) * | 1982-07-30 | 1983-10-30 | Сибирский научно-исследовательский институт нефтяной промышленности | Process for determining organo-phosphoric compaunds containing phosphonic group |
| CN102033011A (en) * | 2010-11-26 | 2011-04-27 | 河北农业大学 | Soil phytic acid extracting solution and application thereof |
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| US5762911A (en) * | 1996-03-05 | 1998-06-09 | The Research Foundation Of State University Of New York | Anti-caries oral compositions |
| US6774288B1 (en) * | 1997-07-07 | 2004-08-10 | Basf Plant Science L.L.C. | Animal feed with low phytic acid, oil, burdened and protein laden grain |
| CA2797972A1 (en) * | 2004-05-20 | 2005-12-01 | Pioneer Hi-Bred International, Inc. | Maize multidrug resistance-associated protein polynucleotides and methods of use |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SU1051415A1 (en) * | 1982-07-30 | 1983-10-30 | Сибирский научно-исследовательский институт нефтяной промышленности | Process for determining organo-phosphoric compaunds containing phosphonic group |
| CN102033011A (en) * | 2010-11-26 | 2011-04-27 | 河北农业大学 | Soil phytic acid extracting solution and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| Journal of Agricultural and Food Chemistry, Vol. 28 (6), 1980, Latta, M., et al., 'A Simple and Rapid Colorimetric Method for Phytate Determination', pp. 1313-1315. * |
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| GB201317332D0 (en) | 2013-11-13 |
| GB2516702B (en) | 2015-09-02 |
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