Notice of Pre-AIA or AIA Status
The present application, filed on or after December 01, 2023, is being examined under the first inventor to file provisions of the AIA .
Claim Status
Claims 1-11 are pending and under examination.
Priority
The present application claims the priority benefit of United States Provisional Application No. 63/429,73 9, filed on December 2, 2022.
Information Disclosure Statement
An Information Disclosure Statement filed on June 12, 2024 is acknowledged and considered.
Drawings
The drawings filed on December 01, 2023 are acknowledged and considered.
Claim Interpretation
Claims 1-11 are direction to a method of producing aflatoxin-contaminated nuts selected from nut species (i.e., almonds. Cashews, hazelnuts, macadamias, peanuts. Pecans. Pistachios, or walnuts) in vitro by preparing a conidial suspension of toxigenic Aspergillus strain (i.e., Aspergillus flavus or Aspergillus parasiticus) inoculating nuts with the suspension, and incubating the inoculated nuts under conditions that allow fungal growth and aflatoxin production.
Abstract Objection
The abstract of the disclosure is objected to because the abstract is a single sentence; and does not provide a concise and complete. A corrected abstract of the disclosure is required and must be presented on a separate sheet, apart from any other text. See MPEP § 608.01(b).
Applicant is reminded of the proper language and format for an abstract of the disclosure.
The abstract should be in narrative form and generally limited to a single paragraph on a separate sheet within the range of 50 to 150 words in length. The abstract should describe the disclosure sufficiently to assist readers in deciding whether there is a need for consulting the full patent text for details.
The language should be clear and concise and should not repeat information given in the title. It should avoid using phrases which can be implied, such as, “The disclosure concerns,” “The disclosure defined by this invention,” “The disclosure describes,” etc. In addition, the form and legal phraseology often used in patent claims, such as “means” and “said,” should be avoided.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or non-obviousness.
Claims 1-3, 5-6, and 9-11 are rejected under 35 U.S.C. 103 as being unpatentable over foreign patent CN1556391A (2004, hereafter “CN1556391A”), in further view of Møller et.al, 2021 (hereafter “Møller”, see Form 892).
CN1556391A teaches a method of producing peanuts in vitro, cultivated with a toxigenic Aspergillus flavus strain (i.e., Aspergillus flavus strain with high yield poison ability [Pg. 4, Summary of Invention, Para. 2]) to cause an artificial infection [Pg. 2, Background Technology, Para. 1]); and used to identify peanut varieties resistant to aflatoxin-contamination (i.e., identifying malicious resistance [Background Technology, Para. 1]). CN1556391A teaches sterilizing and rehydrating peanut seeds (i.e., getting healthy peanut seeds; placing the peanut seeds on a sterile petri dish; spraying the surface of the seeds 1-1.5 minute with 70% alcohol and aqua flushing; and rehydration at 20-30% [Pg. 4, Summary of the Invention, Para. 4, Number 1]); inoculating healthy peanuts with an Aspergillus flavus strain (i.e., cultivating the peanuts for 7 days in an incubator (i.e., inoculation of peanuts by adding 1 milliliter of Aspergillus flavus spores in suspension [Pg. 4, Summary of the Invention, Para. 5, Number 2]); extracting aflatoxin liquid; and determining and calculating the amount of aflatoxin liquid (i.e., adding ammonia mercury solution on fluor spectrophotometer, and measuring fluorescent values to calculate aflatoxin B1 content; and estimate the resistance grade with the typical curve between aflatoxin B1 and the fluorescent value [Abstract; Claim 1; Pg. 4, Summary of Invention, Para. 3]). CN1556391A teaches cultivating after the inoculation in a double dish is placed in plastic casing and adding a cover; adding the suspension 2-3 millimeters deep in the plastic casing; where the interior relative humidity of box is 80-90%; and plastic casing is put into 30 ℃ constant incubator and is cultivated 7 days (Pg. 4, Summary of the Invention, Para. 6, Number 3). CN1556391A teaches after the inoculation, a double dish is placed the plastic casing of adding a cover, add the suspension of a small amount of (water layer thickness 2-3 millimeter) in the plastic casing, guarantee that the relative humidity of sub environment is 80-90%.The constant incubator that plastic casing is put into 30 ℃ was cultivated 7 days (Pg. 5, Embodiment, Para. 3). CN1556391A extracting and determination of toxin concentrations, wherein 10 milliliters of aflatoxin extracts gets filtrated (i.e., 20 microliters through the filter paper filtering of sterilization, add 4 milliliters of ammonia mercury solutions, fully mixing is got 1 milliliter in cuvette, colorimetric on fluor spectrophotometer); and calculating toxin B1 content with calibration curve method (typical curve is gone up at fluor spectrophotometer (LS55) with U.S. Sigma company product aflatoxin B1 standard model and is set up typical curve [Pg. 7, Summary of the Invention, Para. 8, Number 5]). CN1556391A teaches the toxin concentration of high anti-peanut varieties is 5.0 micrograms/below the gram, in anti-kind content of toxins be 5.1-15.0 microgram/gram, the susceptible variety content of toxins is 15.1-40.0 microgram/gram (Pg. 7, Summary of the Invention, Para. 9, Number 6).
CN1556391A does not teach preparing a conidial suspension; decontaminating nuts with hypochlorite; incubating the inoculated nuts at >97% relative humidity (RH) environment, and the inoculated nuts comprising 150 to 300 ppm aflatoxins.
Møller teaches the Preparation of the Conidial Suspension (section subtitle), wherein preparing a conidial suspension using an Aspergillus strain by inoculating it in malt extract agar; incubating it aerobically at 25°C in a BOD incubator for 5 days, at static condition in the dark; collecting conidia by scraping the mycelium from each plate with sterile distilled water containing 0.1% Tween 80; filtering and centrifuging at 11,000 g three consecutive times for 15 min at 4°C; determining the final concentration of conidia in the fungal suspension using a Neubauer chamber; and diluting the conidial suspension to reach a final concentration of 5.0 log10 CFU ml−1 in the growth trials (Pg. 3, “Preparation of the Conidial Suspension”, Col. 1-2, Para. 1).
A person of ordinary skill in the art would have been motivated to combine the methods of CN1556391A (i.e., producing nuts in vitro, cultivated with a toxigenic Aspergillus strain; wherein the peanuts are inoculated, and incubated; Aspergillus mold grows on the nuts; and produces aflatoxins), with the methods of Møller (i.e., standard conidial suspension known in the art using an Aspergillus strain) in order to utilize the conidial suspension of Møller for the inoculum of the peanuts of CN1556391A; and improve the consistency, uniformity, and reproducibility of inoculation of the peanuts with Aspergillus mold and producing aflatoxins as the result. Additionally, both CN1556391A and Møller are directed to the inoculation of Aspergillus for the evaluation of aflatoxin; and Møller teaches that preparing and standardizing conidial suspensions of Aspergillus provides controlled and reproducible fungal exposure; wherein CN1556391A relies on fungal inoculation to assess aflatoxin production. Therefore, it would have been obvious to use Møller’s standardized suspension technique to provide a known and quantifiable inoculum, and ensure consistent infection conditions and predictable aflatoxin production.
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filling date of the claimed invention to combine the methods of CN1556391A and Møller with a reasonable expectation of success because combining the methods would have resulted in an optimized method of producing aflatoxin-contaminated nuts in vitro (i.e., preparing a conidial suspension from a toxigenic Aspergillus strain; inoculating the nuts (peanuts) with the conidial suspension; incubating the inoculated nuts; growing Aspergillus mold, and producing aflatoxins). The combination would have yielded predictable results to one of ordinary skill in the art at the time of the invention (As described MPEP 2143, Section A. Combining Prior Art Elements According to Known Methods to Yield Predictable Results).
Regarding Claim 1: CN1556391A teaches a method producing nuts (i.e., peanuts) in vitro, cultivated with a toxigenic Aspergillus strain (i.e., Aspergillus flavus strain with high yield poison ability to cause an artificial infection); wherein the peanuts are inoculated with the toxigenic Aspergillus flavus strain and incubated; then Aspergillus mold grows and produces aflatoxin. Møller further teaches standard preparation of a conidial suspension known in the art using an Aspergillus strain.
Regarding Claim 2: CN1556391A teaches a method of producing nuts (i.e., peanuts) in vitro, cultivated with a toxigenic Aspergillus strain; wherein the peanuts are inoculated, incubated, and Aspergillus mold grows and produces aflatoxin (previously discussed); wherein the toxigenic Aspergillus strain is an Aspergillus flavus strain (i.e., Aspergillus flavus strain with high yield poison ability to cause an artificial infection). Møller further teaches standard preparation of a conidial suspension known in the art using an Aspergillus strain.
Regarding Claim 3: CN1556391A teaches a method producing nuts (i.e., peanuts) in vitro, cultivated with a toxigenic Aspergillus strain; wherein the peanuts are inoculated, incubated, and Aspergillus mold grows and produces aflatoxin (previously discussed); wherein the method comprising decontamination of the peanuts prior to inoculation; wherein sterilizing and rehydrating the peanut seeds (i.e., getting healthy peanut seeds; placing the peanut seeds on a sterile petri dish; spraying the surface of the seeds 1-1.5 minute with 70% alcohol and aqua flushing; and rehydration at 20-30%). Møller further teaches standard preparation of a conidial suspension known in the art using an Aspergillus strain.
Regarding Claim 5 and 6: CN1556391A teaches a method producing nuts (i.e., peanuts) in vitro, cultivated with a toxigenic Aspergillus strain; wherein the peanuts are inoculated, incubated, and Aspergillus mold grows and produces aflatoxin (previously discussed). Møller further teaches standard preparation of a conidial suspension known in the art using an Aspergillus strain and the conidial suspension reaching a final concentration of 5.0 log10 CFU ml−1 in the growth trials. The concentration disclosed by Møller fails within the claimed range of 104 to 107 CFU/ml (i.e., 1 x 105 CFU/ml). As noted, the concentration of the conidial suspension disclosed by Møller is 5.0 log10 CFU ml−1 in (i.e., equivalent to 1X 105 CFU/ml).
Regarding Claim 9: CN1556391A teaches a method producing nuts (i.e., peanuts) in vitro, cultivated with a toxigenic Aspergillus strain; wherein the peanuts are inoculated, incubated, and Aspergillus mold grows and produces aflatoxin (previously discussed); herein the nuts are selected from almonds, cashews, hazelnuts, macadamias, peanuts, pecans, pistachios, or walnuts. CN1556391A teaches the selection of peanuts). Møller further teaches standard preparation of a conidial suspension known in the art using an Aspergillus strain and the conidial suspension reaching a final concentration of 5.0 log10 CFU ml−1 in the growth trials.
Regarding Claim 10: CN1556391A teaches a method producing nuts (i.e., peanuts) in vitro, cultivated with a toxigenic Aspergillus strain; wherein the peanuts are inoculated, incubated, and Aspergillus mold grows and produces aflatoxin (previously discussed); wherein the inoculated peanuts comprise toxins (i.e., aflatoxins) amounts of 5.1-15.0 microgram/gram, and the peanuts variety susceptible to aflatoxins have toxins content of 15.1-40.0 microgram/gram. It would have been obvious to a person of ordinary skill in the art to know that aflatoxin concentration is a result-effective variable (i.e., a parameter recognized in the art to affect measurable outcomes such as detectability); and would have known that optimizing or adjusting that variable is routine optimization to achieve an intended result. Therefore, the detection of toxins taught by CN1556391A would vary depending on parameters and a skilled artisan would have been motivated to adjust known growth parameters taught by CN1556391A to obtain higher aflatoxin growth concentrations; including concentrations of 150 to 300 ppm for the purposes such as improving detection, testing limits, or ensuring sufficient toxin levels in experimental or contamination models. in order to achieve increased fungal growth and aflatoxin production; wherein increasing the relative number of aflatoxins produced is a routine optimization step. The selection of a particular value or subrange of a known result-effective variable is a matter of routine optimization absent a showing of criticality or unexpected results (see In re Aller). It would have been obvious to arrive at aflatoxin concentration range of 150-300 ppm through routine optimization.
Regarding Claim 11, CN1556391A teaches a method producing nuts (i.e., peanuts) in vitro, cultivated with a toxigenic Aspergillus strain; wherein the peanuts are inoculated, incubated, and Aspergillus mold grows and produces aflatoxin (previously discussed); wherein the incubated, and inoculated nuts (i.e., peanuts) comprise B1 (AFB 1). ). Møller further teaches standard preparation of a conidial suspension known in the art using an Aspergillus strain and the conidial suspension (previously discussed).
Claim 4 is rejected under 35 U.S.C. 103 as being unpatentable over CN1556391A and Møller as applied to Claim 1-3, 5-6, and 9-11 above, and further in view of Natarajan et.al., 1975 (hereafter “Natarajan”, See Form 892).
CN1556391A and Møller render obvious a method of producing nuts (i.e., peanuts) in vitro, comprising preparing a conidial suspension from a toxigenic Aspergillus strain; inoculating the nuts with the conidial suspension; and incubating the inoculated nuts; wherein the Aspergillus mold grows and produces aflatoxin (previously discussed). CN1556391A and Møller are relied upon for the reasons set forth above.
CN1556391A and Møller do not teach decontaminating the nuts comprising contacting with sodium hypochlorite; then rinsing with nuts with sterile water.
Natarajan teaches a method of decontaminating nuts using sodium hypochlorite (NaOCl) as very effective in destroying aflatoxins during the process for the preparation of protein isolates either from raw peanuts or peanut meal (Pg. 1193, Col. 1. Introduction, Para. 2). Natarajan further teaches sodium hypochlorite is a very potent oxidizing agent and, in contact with protein, has a potential for inducing change (Pg. 1193, Col. 1. Introduction, Para. 3).
Although the method of CN1556391A teaches decontaminating the peanuts with 70% alcohol, the method of decontamination taught by Natarajan uses sodium hypochlorite; which is known in the art to effectively destroy aflatoxins prior to inoculation. Therefore, a person of ordinary skill in the art would have been motivated to use both 70% alcohol and sodium hypochlorite as an optimized decontamination step before the inoculation of the peanuts with the toxigenic Aspergillus flavus strain.
A person of ordinary skill in the art would have been motivated to combine the method of CN1556391A (i.e., producing nuts in vitro, cultivated with a toxigenic Aspergillus strain; wherein the peanuts are inoculated, incubated; and Aspergillus mold grows on the nuts and produces aflatoxins), with the method of Møller (i.e., standard preparation of a conidial suspension known in the art using an Aspergillus strain), and Natarajan (i.e., decontaminating nuts using sodium hypochlorite [NaOCl]) in order to utilize a conidial suspension as the inoculum of CN1556391A and improve the consistency, uniformity, and reproducibility of inoculation of the peanuts (e.g., decontaminating the peanuts prior to inoculum with Aspergillus mold); and producing aflatoxins as the result.
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filling date of the claimed invention to combine the methods of CN1556391A, Møller, and Natarajan with a reasonable expectation of success because combining the methods would have resulted in an optimized method of producing aflatoxin-contaminated nuts in vitro (i.e., decontaminating the nuts prior to inoculating; preparing a conidial suspension from a toxigenic Aspergillus strain; inoculating the nuts (peanuts) with the conidial suspension; incubating the inoculated nuts; growing Aspergillus mold, and producing aflatoxins). The combination would have yielded predictable results to one of ordinary skill in the art at the time of the invention (As described MPEP 2143, Section A. Combining Prior Art Elements According to Known Methods to Yield Predictable Results).
Regarding Claim 4: CN1556391A teaches a method producing nuts (i.e., peanuts) in vitro, cultivated with a toxigenic Aspergillus strain; wherein the peanuts are inoculated, incubated, and Aspergillus mold grows and produces aflatoxin (previously discussed); wherein the method comprises decontaminating the peanuts prior to inoculation by sterilizing and rehydrating the peanut seeds (i.e., getting healthy peanut seeds; placing the peanut seeds on a sterile petri dish; spraying the surface of the seeds 1-1.5 minute with 70% alcohol and aqua flushing; and rehydration at 20-30%). Natarajan further teaches a method of decontaminating nuts using sodium hypochlorite [NaOCl]) as an effective way in destroying aflatoxins during the process for the preparation of protein isolates either from raw peanuts or peanut meal. Møller further teaches standard preparation of a conidial suspension known in the art using an Aspergillus strain (previously discussed).
Claim(s) 7-8 are rejected under 35 U.S.C. 103 as being unpatentable over CN1556391A and Møller, as applied to Claims 1-3, 5-6, and 9-11 above; in further in view of Arrus et.al., 2005 (hereafter “Arrus”, see Form 892), and Mannaa et.al, 2018 (hereafter, “Mannaa”, see Form 892).
CN1556391A and Møller render obvious a method of producing nuts (i.e., peanuts) in vitro, comprising preparing a conidial suspension from a toxigenic Aspergillus strain; inoculating the nuts with the conidial suspension; and incubating the inoculated nuts (i.e., at 25°C. to 30°C. for 2 to 10 days and 4 to 10 days); wherein the Aspergillus mold grows and produces aflatoxin (previously discussed). CN1556391A and Møller are relied upon for the reasons set forth above.
CN1556391A and Møller do not teach incubating the inoculated nuts at >97% relative humidity (RH) environment. However, CN1556391A teaches incubating the inoculated nuts at 30°C for 7 days.
Arrus teaches effects of relative humidity (RH.; 75%, 80%, 85%, 97%) and temperature (10, 13, 15, 25, 30 °C) on aflatoxin production in previously dried (3.5% moisture content) Brazil nuts (Abstract). Arrus further teaches the maximum concentrations of total aflatoxin and B1 were detected in nuts stored at 97% RH. and at temperatures of 25–30 °C.
Mannaa teaches relative humidities (RHs; 12, 44, 76, and 98%) on populations of predominant grain fungi (e.g., Aspergillus flavus) and populations of all the tested fungi in inoculated rice grains were significantly enhanced by both increased temperature and RH (Abstract).
CN1556391A and Møller teach a method of inoculating nuts with a toxigenic Aspergillus strain to produce aflatoxin. Arrus teaches that relative humidity (RH) directly affects Aspergillus growth and aflatoxin production in nuts; and teaches that higher RH (i.e., 97%) result in increased aflatoxin levels. Mannaa further teaches that increasing RH to 98% (e.g., >97% RH) enhances Aspergillus growth (increasing aflatoxin production). CN1556391A, Møller, Arrus, and Mannaa are directed to the increased growth of Aspergillus under parameters and conditions. Therefore, it is well understood in the art that Aspergillus growth, which leads to aflatoxin production increases with increasing incubation temperature and RH, and temperature and RH is a parameter that can be adjusted to promote Aspergillus growth (and aflatoxin production). A person of ordinary skill in the art would have been motivated to modify the incubation conditions of CN1556391A and Møller to include RH >97%, as taught by Mannaa to increase Aspergillus growth and aflatoxin production in nuts (as taught by Arrus), with a reasonable expectation of success because the relationship between increased RH and increased Aspergillus growth is well established.
A person of ordinary skill in the art would have been motivated to combine the method of CN1556391A (i.e., producing nuts in vitro, cultivated with a toxigenic Aspergillus strain; wherein the peanuts are inoculated, incubated at 25°C. to 30°C. for 2 to 10 days and 4 to 10 days; Aspergillus mold grows; and produces aflatoxin), with the method of Møller (i.e., standard preparation of a conidial suspension known in the art using an Aspergillus strain); Arrus (i.e., increased Aspergillus growth and aflatoxin production in nuts stored at a higher H [97% RH]); and Mannaa (i.e., increased Aspergillus growth at a higher RH parameter >97%) in order to utilize a conidial suspension as the inoculum of CN1556391A and improve the consistency, uniformity, and reproducibility of inoculation of nuts (e.g., incubated at 25°C. to 30°C. for 2 to 10 days and 4 to 10 days; and at >97% RH) and producing aflatoxins as the result.
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filling date of the claimed invention to combine the methods of CN1556391A, Møller, Arrus, and Mannaa with a reasonable expectation of success because combining the methods would have resulted in an optimized method of producing aflatoxin-contaminated nuts in vitro (i.e., decontaminating the nuts prior to inoculating; preparing a conidial suspension from a toxigenic Aspergillus strain; inoculating the nuts (peanuts) with the conidial suspension; incubating the inoculated nuts (i.e., at 25°C. to 30°C. for 2 to 10 days and 4 to 10 days and stored at >97% RH); growing Aspergillus mold, and producing aflatoxins. The combination would have yielded predictable results to one of ordinary skill in the art at the time of the invention (As described MPEP 2143, Section A. Combining Prior Art Elements According to Known Methods to Yield Predictable Results).
Regarding Claim 7 and 8: CN1556391A and Møller teach a method producing nuts (i.e., peanuts) in vitro, cultivated with a toxigenic Aspergillus strain (i.e., inoculating the nuts with the conidial suspension; wherein the Aspergillus mold grows and produces aflatoxin (previously discussed); wherein the incubation of the inoculated peanuts comprises putting the peanuts into 30 ℃ constant incubator and is cultivated for 7 days (within the range of 2 to 10 days and 4 to 7 days). Arrus further teaches maximum concentrations of total aflatoxin and B1 were detected in nuts stored at 97% (RH) and at temperatures of 25–30 °C, and Mannaa further teaches >97% RH (i.e., 98% RH) for growing Aspergillus mold. A person of ordinary skill in the art would have been motivated to increase the relative humidity (RH) of the incubation environment taught by CN1556391A to a higher RH when incubating nuts, as taught by Arrus; and RH >97%, as taught by Mannaa in order to achieve increased fungal growth, aflatoxin production, and routine optimization of the incubation environment conditions.
Conclusion
No claim is allowed.
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/ENOSAKHARE ERHUNMWUNSEE/Examiner, Art Unit 1651
/MELENIE L GORDON/ Supervisory Patent Examiner, Art Unit 1651
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