Reducing aflatoxin content in nuts by ultraviolet and / or oxidation treatment

Abstract

The present invention provides a method of reducing aflatoxin contamination in one or more nuts and reducing the content of Aspergillus flavus and / or Aspergillus parasiticus resulting in aflatoxin contamination in one or more nuts. In one embodiment, nuts are treated with ultraviolet (UV) rays, ozone and peroxide, or a combination thereof. In one embodiment, nuts are treated with UV rays and ozone. In one embodiment, nuts are treated with UV rays and peroxide. In one embodiment, nuts are treated with ozone and peroxide.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 429,739, filed on December 2, 2022, the entire contents of which are hereby incorporated by reference into this application.

[0003] field

[0004] The present invention relates to a method for reducing aflatoxin contamination in foods such as nuts. Technical Field

[0005] Aflatoxins are a class of structurally related fungi. Produced by specific Aspergillus species, aflatoxins are toxic, mutagenic, and / or carcinogenic. Aspergillus flavus and Aspergillus parasiticus are the main toxin-producing species. A. flavus is not host-specific and can infect a variety of food crops, while A. parasiticus has stronger host specificity and may contaminate peanuts in particular. See, e.g., Jallow et al., 2021, Compr Rev Food Sci Food Saf. 20:2332–2381.

[0006] Aflatoxin contamination and fungal infestations are present in agricultural products and foods, such as nuts, cocoa, and spices, and therefore pose a significant consumer safety concern. Supply chain monitoring measures can reduce the risk of consumer exposure, but they can also result in significant economic losses due to the need to destroy contaminated food. This invention aims to provide a solution to this problem. Summary of the Invention

[0007] The present invention generally relates to a method for reducing aflatoxin contamination in one or more nuts, and / or reducing the content of Aspergillus flavus and / or Aspergillus parasiticus that contribute to aflatoxin contamination in one or more nuts. The method of the present invention comprises contacting the one or more nuts with one or more treatments, thereby reducing the presence of aflatoxin and / or reducing the content of Aspergillus flavus and / or Aspergillus parasiticus that contribute to the presence of aflatoxin, relative to the one or more nuts prior to exposure to the one or more treatments. The method of the present invention comprises contacting the one or more nuts with one or more treatments, thereby reducing the presence of aflatoxin, relative to the one or more nuts prior to exposure to the one or more treatments. The method of the present invention further comprises contacting the nuts with one or more oxidizing agents, such as ultraviolet (UV) light, ozone (O3), peroxides (e.g., hydrogen peroxide, H2O2), electrolytically oxidized water (EOW), pulsed electric fields (PEF), supercritical carbon dioxide, cold plasma, organic acid cleaning, and combinations thereof. For example, a treatment method may include exposing one or more nuts to ultraviolet light and exposing one or more nuts to one or more oxidizing compounds, such as ozone or peroxides, for example, by gas exposure and / or exposure in a solvent or solution, such as an aqueous ozone solution. The ultraviolet light and the oxidizing compounds may be applied simultaneously or sequentially in any order.

[0008] BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The accompanying drawings are incorporated in and constitute a part of this specification and serve to illustrate the various methods and compositions disclosed herein.

[0010] Figure 1 A, 1B and 1C show hazelnuts contaminated with Aspergillus niger ( Figure 1 Data for nuts (A) and almonds (B) after exposure to ultraviolet (UV) light for different times (1 hour, 4 hours, and 36 hours) at a fixed power. "Native control"—nuts not contaminated with Aspergillus. "After inoculation and incubation"—nuts inoculated with Aspergillus and then incubated to obtain contaminated nuts. Figure 1 C shows the final summary of data for hazelnuts and almonds. See Example 1c.

[0011] Figure 2 A and 2B show data for hazelnuts and almonds contaminated with Aspergillus spp. after treatment with either gaseous ozone (hazelnuts) or aqueous ozone (almonds). Two doses were tested for each ozone treatment method. Aspergillus cell counts ( Figure 2 A) and aflatoxins ( Figure 2 B; the sum of aflatoxins B1 and B2), a treated control group was also evaluated. See Example 2c.

[0012] Figure 3 Data are shown for the growth and aflatoxin production of three different Aspergillus strains on clean hazelnuts, obtained under two different culture conditions and six time points. See Example 10. DETAILED DESCRIPTION

[0013] Definitions and Abbreviations

[0014] Unless defined otherwise, all technical and scientific terms used in this specification generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0015] As used herein, the articles "a" and "an" are used to refer to one or more (ie, at least one) of the grammatical objects modified by the article. For example, "an element" means one element or a plurality of elements.

[0016] As used herein, "about" when referring to a measurable value (eg, an amount, a period of time, etc.) refers to a deviation of ±10% from the specified value, as such deviations are appropriate for practicing the methods of the invention.

[0017] In this article, the term "aflatoxins" refers to a class of structurally related mycotoxins produced by certain fungi, such as Aspergillus flavus and Aspergillus parasiticus. Aflatoxins may be toxic, mutagenic, and / or carcinogenic. The B-series (aflatoxins B1 and B2), G-series (aflatoxins G1 and G2), and M-series aflatoxins are of primary concern and are regulated under food and agricultural product safety regulations. Aflatoxin B1 (AFB1) is the most carcinogenic of the aflatoxins.

[0018] In this document, the terms “reduction of aflatoxin contamination,” “reduction of aflatoxin contamination,” and “aflatoxin reduction” refer to the reduction of detectable levels of aflatoxins (e.g., AFB1) in a food (e.g., nuts or a variety of nuts). These terms encompass decontamination, detoxification, and a combination of decontamination and detoxification. Decontamination refers to the physical removal of aflatoxins and / or the removal of Aspergillus flavus and Aspergillus parasiticus. Detoxification refers to the degradation of aflatoxins (e.g., AFB1) and / or Aspergillus flavus and Aspergillus parasiticus.

[0019] Range: Within the entire scope of the present invention, various aspects of the present invention may be presented in the form of a range. It should be understood that the description in the form of a range is only for convenience and brevity and should not be interpreted as limiting the scope of the present invention. Therefore, the description of a range should be regarded as having explicitly disclosed all possible sub-ranges within the range and each numerical value within the range. For example, the description of the range "from 1 to 6" should be regarded as having explicitly disclosed the following sub-ranges: from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as all specific numerical values ​​within the range, such as: 1, 2, 2.7, 3, 4, 5, 5.3 and 6. This principle applies to any width of the range.

[0020] It is to be understood that any and all or partial integers recited in any range specified herein are encompassed herein.

[0021] As shown in the compositions of matter and methods disclosed herein, embodiments of the present invention, in another aspect, comprise the components and / or steps disclosed herein. In another aspect, embodiments of the present invention consist essentially of the components and / or steps disclosed herein. In another aspect, embodiments of the present invention consist of the components and / or steps disclosed herein.

[0022] Finally, unless otherwise indicated herein or clearly contradicted by context, the steps of all methods described herein may be performed in any suitable order. Any and all examples or exemplary language (e.g., "for example") provided herein are intended solely to better illustrate the present invention and do not constitute a limitation on the scope of the present invention unless otherwise provided in the claims. Numerous modifications and adaptations will be apparent to those skilled in the art without departing from the spirit and scope of the present invention.

[0023] describe

[0024] The embodiments of the present invention are described below. However, it should be clearly pointed out that the present invention is not limited to these embodiments, but is intended to include modifications and equivalent variations obvious to those skilled in the art.

[0025] The present invention generally relates to a method for reducing aflatoxin contamination in one or more nuts and / or reducing the level of Aspergillus flavus and / or Aspergillus parasiticus that cause aflatoxin contamination in one or more nuts. In certain aspects of the method, the method comprises contacting the one or more nuts with one or more treatments, thereby reducing the level of aflatoxin and / or reducing the presence of Aspergillus flavus and / or Aspergillus parasiticus that cause aflatoxin contamination, compared to the one or more nuts prior to contact with the one or more treatments.

[0026] The degree of aflatoxin contamination varies depending on the type of nut. In addition, for the same type of nut, the degree of aflatoxin contamination may also vary depending on the source of the nut. For example, a global assessment of aflatoxin contamination in various nuts from Asia, Africa, Europe and South America found that the average concentration of total aflatoxins (the sum of AFB1, AFB2, AFG1 and AFG2) in peanuts was 40.87 ppb, ranging from 0 ppb (not detected) to 530 ppb (Ebrahimi et al., The prevalence of aflatoxins in different nut samples: A global systematic review and probabilistic risk assessment, AIMS Agriculture and Food, 2022, 7(1):130-148). Similarly, the average concentration of total aflatoxins in pistachios was 37.52 ppb, ranging from 0 ppb (not detected) to 245.6 ppb. The average concentration of total aflatoxins in almonds was 3.54 ppb, ranging from 0 ppb (not detected) to 32.9 ppb; the average concentration of total aflatoxins in hazelnuts was 17.33 ppb, ranging from 0.2 ppb to 124 ppb. Different countries have different allowable standards for aflatoxin contamination in tree nuts. The EU standards are the most stringent. For example, the maximum allowable content of aflatoxins (the sum of AFB1, AFB2, AFG1 and AFG2) in peanuts intended for direct human consumption or as food ingredients is 4 ppb. The maximum allowable content of aflatoxins in almonds, pistachios, hazelnuts and Brazil nuts intended for direct human consumption or as food ingredients is 10 ppb.

[0027] In certain aspects, one or more nuts having at least about 300 ppb aflatoxins, and / or the presence of Aspergillus flavus and / or Aspergillus parasiticus at levels resulting in an aflatoxin content of at least about 300 ppb, are subjected to at least one treatment to reduce the aflatoxin content to less than 300 ppb, and / or to reduce the level of Aspergillus flavus and / or Aspergillus parasiticus to a level of less than 300 ppb aflatoxin. In certain embodiments, the aflatoxin content is reduced by at least one log (e.g., to 30 ppb), or by at least two logs (e.g., to 3 ppb). In certain aspects, one or more nuts having at least about 200 ppb of aflatoxins, and / or the presence of Aspergillus flavus and / or Aspergillus parasiticus at levels resulting in an aflatoxin content of at least about 200 ppb, are subjected to at least one treatment to reduce the aflatoxin content to less than 200 ppb, and / or to reduce the level of Aspergillus flavus and / or Aspergillus parasiticus to a level of less than 200 ppb of aflatoxins. In certain embodiments, the aflatoxin content is reduced by at least one log (e.g., to 20 ppb), or by at least two logs (e.g., to 2 ppb).

[0028] In certain embodiments, the methods of the present invention can reduce the level of aflatoxins by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%.

[0029] In certain embodiments, the methods of the present invention can reduce the level of aflatoxins by at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90%.

[0030] In certain embodiments, the methods of the present invention can reduce the level of aflatoxins by at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0031] In certain embodiments, the methods of the present invention can reduce the level of aflatoxins by at least about 0.5 log, at least about 1 log, at least about 1.5 logs, at least about 2 logs, or at least about 2.5 logs.

[0032] In certain embodiments, the methods of the present invention reduce the level of aflatoxins to less than about 200 ppb, less than about 190 ppb, less than about 180 ppb, less than about 170 ppb, less than about 160 ppb, less than about 150 ppb, less than about 140 ppb, less than about 130 ppb, less than about 120 ppb, less than about 110 ppb, less than about 100 ppb, or less than about 10 ppb.

[0033] In certain embodiments, the methods of the present invention reduce the level of aflatoxins to less than about 95 ppb, less than about 90 ppb, less than about 85 ppb, less than about 80 ppb, less than about 75 ppb, less than about 70 ppb, less than about 65 ppb, less than about 60 ppb, less than about 55 ppb, or less than about 50 ppb.

[0034] In certain embodiments, the methods of the present invention reduce the level of aflatoxins to less than about 45 ppb, less than about 40 ppb, less than about 35 ppb, less than about 30 ppb, less than about 25 ppb, less than about 20 ppb, less than about 15 ppb, less than about 10 ppb, less than about 5 ppb, less than about 4 ppb, or 2 ppb or less.

[0035] As used herein, "nuts" refers to edible nuts. Examples of edible nuts include, but are not limited to, almonds, cashews, hazelnuts, macadamia nuts, peanuts, pecans, pistachios, and walnuts. In the methods of the present invention, the nuts used for processing may be in their shells (in-shell nuts) or without their shells (shelled nuts). Cracked shelled nuts, including but not limited to chopped nuts, sliced ​​nuts, nut butters, and nut pastes, may also be processed in the methods of the present invention.

[0036] Various aspects of the present invention are designed to address aflatoxin contamination while minimizing the impact on the integrity of the underlying food product. For example, when one or more aflatoxin reduction processes are applied to nut products, the nuts are not cooked by the reduction techniques, and the levels of residual chemicals are not high enough to prevent the nuts from being sold in the same product category or negatively impact the consumer experience.

[0037] The method of the present invention comprises subjecting one or more nuts to one or more treatments to reduce the content of aflatoxins and / or reduce the presence of Aspergillus flavus and / or Aspergillus parasiticus, which cause aflatoxin contamination. Exemplary treatment methods include contacting one or more nuts with an oxidizing agent, such as ultraviolet (UV) light, ozone (O3) and / or peroxides (e.g., hydrogen peroxide H2O2), electrolytic oxidation of water (EOW), pulsed electric field (PEF), cold plasma, supercritical fluid extraction (SFE) using supercritical carbon dioxide (SC CO2), one or more chemical cleanings, and combinations thereof. Other potential oxidizing agents include, for example, hypochlorites such as sodium chlorite, or oxidizing gases such as oxygen (O2) or nitrous oxide. In one aspect, the nuts can receive multiple treatments, either sequentially or in parallel.

[0038] Ultraviolet (UV) light

[0039] In one embodiment of the present method, one or more nuts having at least about 300 ppb aflatoxins, and / or the presence of Aspergillus flavus and / or Aspergillus parasiticus at levels resulting in an aflatoxin content of at least about 300 ppb, are exposed to ultraviolet (UV) light to reduce the aflatoxin content to less than 300 ppb, and / or reduce the level of Aspergillus flavus and / or Aspergillus parasiticus to a level of less than about 300 ppb aflatoxin. In certain embodiments, the aflatoxin content is reduced by at least half a log (e.g., to about 95 ppb), at least one log (e.g., to about 30 ppb), at least 1.5 logs (e.g., to about 9.5 ppb), or at least two logs (e.g., to about 3 ppb). Without being bound by theory, the inventors believe that high-energy photons from UV light have sufficient energy to break chemical bonds within organic molecules. In the case of aflatoxins, this may result in the hydrogenation of the furan ring (the site of aflatoxin toxicity). Another potential advantage of using UV light is the generation of other reactive species, such as oxygen free radicals, which can then react with and degrade aflatoxins and their primary metabolites.

[0040] The ultraviolet spectrum covers a wavelength range of about 10 nm to about 400 nm. The ultraviolet spectrum is divided into three regions: UV-A, UV-B, and UV-C. UV-A light covers a wavelength of about 320 nm to about 400 nm. UV-B light covers a wavelength of about 280 nm to about 320 nm, and UV-C light covers a wavelength of about 100 nm to about 280 nm. UV treatment of nuts can be performed using any of the three UV regions mentioned above. UV-C light may oxidize nut oils, which may adversely affect taste. UV treatment can be monochromatic (essentially a single wavelength) or polychromatic (a range of wavelengths). In certain embodiments, the UV treatment is UV-A light. In certain embodiments, ultraviolet A (UV-A) light treatment is monochromatic light with a wavelength of about 350 nm or about 365 nm. In certain embodiments, the UV-A light treatment is polychromatic light, for example, from about 315 nm to about 400 nm or from about 345 nm to about 400 nm. In certain embodiments, the ultraviolet light treatment is polychromatic, such as from about 140 nm to about 365 nm, from about 350 nm to about 365 nm, or from about 140 nm to about 350 nm. In certain embodiments, the ultraviolet light treatment is UV-C light. In certain embodiments, the UV-C light treatment is monochromatic, about 256 nm (e.g., about 256 nm, or about 251-261 nm, or about 255-257 nm), about 254 nm (e.g., about 254 nm, or about 249-259 nm, or about 253-255 nm), or about 142 nm (e.g., about 142 nm, or about 137-147 nm, or about 141-143 nm). In certain embodiments, the UV-C light treatment is less than 256 nm, less than 254 nm, less than 225 nm, less than 190 nm, or less than 143 nm. In a combined treatment, when the second treatment may be adversely affected by higher wavelengths, such as ozone, the UV-C light should be below 190 nm, such as about 186 nm or 142 nm, to minimize the risk that ozone may be inactivated at higher wavelengths. The UV treatment may be from about 15 minutes to about 4 hours, or from about 15 minutes to about 1 hour, or from about 30 minutes to about 1 hour, or from about 30 minutes to about 2 hours, such as about, or not more than, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, or 4 hours. The UV treatment may also exceed 4 hours, such as, but not limited to, about 6 hours, about 12 hours, about 24 hours, or more. However, extending the UV treatment time may result in the production of additional aflatoxins during the treatment, thereby reducing the overall treatment effectiveness. In certain embodiments, rotating the target (nuts) during the UV treatment process may expose more surface area to the treatment, thereby improving the treatment effectiveness. For example, rotating the nuts during the UV exposure process may be achieved by multiple passes on a multi-head conveyor. The UV treatment may also be carried out in water, which may provide an additional source of active substances.

[0041] Example 1a

[0042] Peanuts were collected and their aflatoxin content was determined by, for example, high performance liquid chromatography (HPLC), and the results showed an average aflatoxin content of at least about 300 ppb. Peanuts were unshelled. One kilogram of unshelled peanuts was subjected to a 2.75 mW / cm 2 The peanuts were irradiated with UV-A light for 30 or 60 minutes. The UV-A light was polychromatic (345 to 400 nm) with a peak output wavelength of 365 nm. The aflatoxin content of the peanuts after UV-A treatment was measured, for example, by high-performance liquid chromatography (HPLC). The results showed that the aflatoxin content was reduced by at least one log from 300 ppb (e.g., to approximately 30 ppb or less) for both 30 and 60 minutes of treatment. By adjusting the treatment conditions, a reduction of approximately two logs in aflatoxin content can be achieved.

[0043] Example 1b

[0044] The hazelnuts were collected and their aflatoxin content was determined by, for example, high performance liquid chromatography (HPLC) to determine an average aflatoxin content of at least about 200 ppb. The hazelnuts were in shell. One kilogram of shelled hazelnuts was subjected to a 2.75 mW / cm 2 The hazelnuts were irradiated with polychromatic UV-A light (wavelength range 345 nm to 400 nm) for 30 minutes or 60 minutes. The UV-A light was polychromatic (wavelength range 345 nm to 400 nm) with a peak output wavelength of 365 nm. High-performance liquid chromatography (HPLC) was used to measure the aflatoxin content of the hazelnuts after UV-A treatment. The results showed that the aflatoxin content was reduced by at least one log from 200 ppb to, for example, about 20 ppb or less, for both 30 and 60-minute treatments.

[0045] Example 1c

[0046] This example investigates the effects of ultraviolet (UV) irradiation on two tree nuts—hazelnuts and almonds. The nuts were first inoculated with Aspergillus (NRRL catalog number NRRL 500) and then cultured to prepare aflatoxin-contaminated test nuts. The experiment included a negative control (uncontaminated nuts) and a positive control (contaminated nuts, treated as closely as possible with the mitigation protocol, but excluding the active step of UV irradiation). The nuts were tested in triplicate. The largest possible sample of nuts (>30 grams) was used for homogenization. The samples were then spotted in triplicate on a total aflatoxin ELISA plate to maximize reading reliability.

[0047] The UV treatment was performed at a fixed wavelength of 256 nm. Therefore, the parameter investigated was the irradiation time. The treatment times tested were: 1 hour, 4 hours, and 36 hours. Within a given irradiation period, the nuts were mixed four times to improve irradiation uniformity. The data for a single replicate experiment are shown in Figure 2. Figure 1 A(hazelnut) and Figure 1 B (almonds). The final data for hazelnuts and almonds are as follows Figure 1 C. This treatment was more effective for hazelnuts than for almonds, but the difference was small. Aflatoxin levels did not show a statistically significant reduction. However, there was significant noise in the results. The formation of other aflatoxins during and after treatment is believed to be a possible source of this noise.

[0048] ozone

[0049] In another embodiment of the disclosed method, one or more nuts having at least about 300 ppb aflatoxins, and / or the presence of Aspergillus flavus and / or Aspergillus parasiticus at levels resulting in an aflatoxin content of at least about 300 ppb, are contacted with ozone (O3) to reduce the aflatoxin content to less than 300 ppb, and / or reduce the level of Aspergillus flavus and / or Aspergillus parasiticus to a level of less than about 300 ppb aflatoxin. In certain embodiments, the aflatoxin content is reduced by at least half a log (e.g., to about 95 ppb), at least one log (e.g., to about 30 ppb), at least 1.5 logs (e.g., to about 9.5 ppb), or at least two logs (e.g., to about 3 ppb).

[0050] Ozone is a powerful oxidant. Ozone treatment can be performed in gaseous or liquid form (ozonated water). Advantageously, gaseous ozone has a half-life of several hours. Liquid ozone contains other substances that can target aflatoxins and also provide a cleaning effect. In certain embodiments, ozone treatment uses gaseous ozone.

[0051] The ozone dosage can be from about 3 mg / l to about 300 mg / l, from about 3 mg / l to about 300 mg / l, from about 3 mg / l to about 250 mg / l, from about 3 mg / l to about 240 mg / l, from about 3 mg / l to about 200 mg / l, from about 3 mg / l to about 150 mg / l, from about 3 mg / l to about 125 mg / l, from about 3 mg / l to about 120 mg / l, from about 3 mg / l to about 100 mg / l, from about 3 mg / l to about 50 mg / l, from about 3 mg / l to about 40 mg / l, from about 3 mg / l to about 20 mg / l, from about 3 mg / l to about 15 mg / l, from about 3 mg / l to about 10 mg / l, from about 3 mg / l to about 7.5 mg / l, or from about 3 mg / l to about 6 mg / l.

[0052] The duration of the ozone treatment can be from about 10 minutes to about 4 days, from about 10 minutes to about 3 days, from about 10 minutes to about 2 days, from about 10 minutes to about 24 hours, from about 10 minutes to about 12 hours, from about 10 minutes to about 6 hours, from about 10 minutes to about 120 minutes, from about 10 minutes to about 60 minutes, from about 10 minutes to about 50 minutes, from about 10 minutes to about 40 minutes, from about 10 minutes to about 30 minutes, from about 10 minutes to about 20 minutes, or from about 10 minutes to about 15 minutes. The ozone treatment can be carried out at a temperature of about 18°C ​​to about 30°C, such as about 20°C, about 22°C, about 25°C, about 27°C, about 30°C, or about 20°C to about 22°C, about 20°C to about 25°C, and about 22°C to about 27°C. In certain embodiments, the nuts to be ozone treated have a moisture content of about 3% to about 8%.

[0053] In certain embodiments, ozone treatment uses gaseous ozone. The dosage of gaseous ozone can be about 120 g / m 3 (grams per cubic meter = mg / l) to 250g / m 3 , about 160g / m 3 Up to 240g / m 3 , about 160g / m 3 , or about 240g / m 3 In one embodiment, the treatment uses about 160 g / m 3 In one embodiment, the treatment is about 240g / m 3 of gaseous ozone for 60 minutes.

[0054] In certain embodiments, ozone treatment uses an aqueous ozone solution. The dosage of the aqueous ozone solution may be about 10 g / m 3 (grams per cubic meter = mg / l) to 50g / m 3 , about 20g / m 3 Up to 40g / m3 , about 20g / m 3 , or about 40g / m 3 In one embodiment, the treatment uses about 20 g / m 3 In another embodiment, the treatment is carried out using an ozone solution of about 40 g / m 3 In some embodiments, the ozone treatment may include both gaseous ozone and ozone delivered via a liquid medium, for example, the nuts may be first exposed to gaseous ozone and then treated with an aqueous ozone solution, or vice versa.

[0055] Example 2a

[0056] Hazelnuts are collected and their aflatoxin content is measured, for example, by high-performance liquid chromatography (HPLC), to determine an average level of at least about 300 ppb. The moisture content of the hazelnuts is measured and found to be approximately 3% to 8%. The hazelnuts are in-shell. One kilogram of the in-shell hazelnuts is treated with 6 mg / L of gaseous ozone for approximately 30 minutes. The aflatoxin content of the ozone-treated hazelnuts is measured, for example, by high-performance liquid chromatography (HPLC), and the results show a reduction of at least one log from 300 ppb, for example, to about 30 ppb or less.

[0057] Example 2b

[0058] Pecans are collected and their aflatoxin content is measured, for example, by high performance liquid chromatography (HPLC), to determine an average aflatoxin content of at least about 200 ppb. The moisture content of the pecans is measured and determined to be approximately 3% to 8%. The pecans are in-shell. One kilogram of the in-shell pecans is treated with 6 mg / l of gaseous ozone for approximately 30 minutes. The aflatoxin content of the ozone-treated pecans is measured, for example, by high performance liquid chromatography (HPLC), to determine a reduction of at least one log from 200 ppb, for example, to about 20 ppb or less.

[0059] Example 2c

[0060] This example studies the effect of ozone on two types of nuts - hazelnuts and almonds. Microbial growth on nuts requires water activity. Therefore, before inoculation, the water activity of the test nuts was determined. If the water activity was too low, the test nuts were placed in a humid environment at 30°C to ensure a water activity of at least 0.6. The nuts were inoculated with Aspergillus (IMI 124931) and then cultured to prepare aflatoxin-contaminated test nuts. Three test batches were prepared for each type of nut. The treatment was carried out at room temperature (about 22°C). Gaseous ozone was applied in the form of high concentration, low temperature (cold) gaseous ozone. Hazelnuts were inoculated at 240 g / m 3 Gaseous ozone treatment was performed for 60 minutes at an ozone dose of 160 g / m3 The ozone dosage was 20g / m 3 ozone solution treatment for 20 minutes at an ozone dose of 40g / m 3 The ozone aqueous solution treatment was carried out for 15 minutes at an ozone dose of 1.5 wt %. The weight gain was monitored during the aqueous solution treatment; when calculating the treatment effect, the average weight gain of 9.5% was used to calculate the correct result.

[0061] The effect of ozone treatment on reducing Aspergillus was evaluated by measuring the number of Aspergillus cells. Figure 2 As shown in Figure A. Data include Aspergillus cell counts before and after ozone treatment (tr) and the log reduction achieved. Results are the average of three replicate analyses for each batch. For aqueous treatments, results were corrected for weight gain so that the data can be directly compared to pre-treatment values. Both gaseous ozone and aqueous ozone solutions reduced Aspergillus. For both gaseous ozone and aqueous ozone, the reduction effect was greater with higher ozone concentrations under shorter exposure times. Under the conditions tested, gaseous ozone was more effective in reducing Aspergillus cell counts.

[0062] In these experiments, colony counts of other molds (other bacterial groups) were also determined. These data are shown in Table 1 and indicate that the treatment was also effective in reducing the numbers of other molds.

[0063] Table 1 - Data on other molds treated with ozone

[0064]

[0065] Data on aflatoxin mitigation such as Figure 2 Figure B. Aflatoxin B1 and B2 levels were measured before and after ozone treatment using 100 g of ground samples. For the aqueous solution treatment, the results were corrected for weight gain so that the data could be directly compared with pre-treatment values. Both aqueous ozone treatments resulted in a reduction in aflatoxin levels, but this was only effective in one of the three batches of nuts. Gaseous ozone treatment significantly reduced aflatoxin levels in all three batches at both doses.

[0066] peroxide

[0067] In another embodiment of the method, one or more nuts having at least about 300 ppb aflatoxins, and / or the presence of Aspergillus flavus and / or Aspergillus parasiticus at levels resulting in an aflatoxin content of at least about 300 ppb, are contacted with a peroxide to reduce the aflatoxin content to less than 300 ppb and / or reduce the level of Aspergillus flavus and / or Aspergillus parasiticus to a level of less than about 300 ppb aflatoxin. In certain embodiments, the aflatoxin content is reduced by at least half a log (e.g., to about 95 ppb), at least one log (e.g., to about 30 ppb), at least 1.5 logs (e.g., to about 9.5 ppb), or at least two logs (e.g., to about 3 ppb).

[0068] In the method of one embodiment of the present invention, the peroxide concentration may range from about 0.1% to about 30% hydrogen peroxide (H2O2), about 0.5% to about 25% H2O2, about 1% to about 20% H2O2, about 10% to about 25% H2O2, and any values ​​and / or subranges therebetween, such as about 1%, about 5%, about 10%, about 20%, and about 30%. The treatment may be carried out at a temperature of about 20°C to about 50°C for a duration of about 1 hour (hr) to about 24 hours, such as about or not more than 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours, or 12 hours. For short treatment times, the hydrogen peroxide concentration should be at a higher level. Lower concentration H2O2 treatments may be effective at higher temperatures and / or longer contact times.

[0069] Alternatively, peroxidase and / or catalase (which inactivates hydrogen peroxide) can be inactivated prior to treatment, for example by subjecting the nuts to a short heat treatment, for example, at about 140° C. for about 10 minutes. Such treatment can achieve effective hydrogen peroxide treatment at lower concentrations of hydrogen peroxide and / or shorter treatment times.

[0070] Example 3a

[0071] Hazelnuts are collected and their aflatoxin content is measured, for example, by high-performance liquid chromatography (HPLC), to determine an average aflatoxin content of at least about 200 ppb. The hazelnuts are in their shells. One kilogram of in-shell hazelnuts is treated with 30% (30 g / gh) hydrogen peroxide (H2O2) (approximately 2 ml H2O2 per gram of hazelnuts) at 50°C for 4 hours. For example, the aflatoxin content of the hazelnuts after H2O2 treatment is measured by HPLC to determine a reduction of at least half a log from 200 ppb (e.g., to about 63 ppb or less).

[0072] Example 3b

[0073] Almonds are collected and their aflatoxin content is measured, for example, by high-performance liquid chromatography (HPLC), to determine an average aflatoxin content of at least about 100 ppb. The almonds are in-shell. One kilogram of in-shell almonds is treated with 30% (30 g / gh) hydrogen peroxide (H2O2) (approximately 2 ml H2O2 per gram of almonds) at 50°C for 4 hours. The aflatoxin content of the H2O2-treated almonds is measured, for example, by high-performance liquid chromatography (HPLC), to determine a reduction of at least one log from 100 ppb (e.g., to about 10 ppb or less).

[0074] Ozone and peroxides

[0075] In another embodiment of the method, one or more nuts having at least about 300 ppb aflatoxins, and / or the presence of Aspergillus flavus and / or Aspergillus parasiticus at levels resulting in an aflatoxin content of at least about 300 ppb, are contacted with ozone (O3) and a peroxide (e.g., H2O2) to reduce the aflatoxin content to less than 300 ppb and / or reduce the level of Aspergillus flavus and / or Aspergillus parasiticus to a level of less than about 300 ppb aflatoxin. In certain embodiments, the aflatoxin content is reduced by at least half a log (e.g., to about 95 ppb), at least one log (e.g., to about 30 ppb), at least 1.5 logs (e.g., to about 9.5 ppb), or at least two logs (e.g., to about 3 ppb).

[0076] In certain embodiments of the combined treatment, the peroxide concentration may range from about 0.1% to about 30% hydrogen peroxide, H2O2, about 0.5% to about 25% H2O2, about 1% to about 20% H2O2, about 10% to about 25% H2O2, and any intermediate values ​​and / or subranges, such as about 1%, about 5%, about 10%, about 20%, and about 30%. The treatment may be carried out at a temperature of about 20°C to about 50°C. The treatment may be carried out for about 1 hour (hr) to about 24 hours, such as about or not more than 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours, or 12 hours. For short-term treatments, the concentration of hydrogen peroxide should be at a higher level. Lower concentration H2O2 treatments may be effective at higher temperatures and / or longer contact times.

[0077] The ozone dosage may range from about 3 mg / l to about 300 mg / l, from about 3 mg / l to about 300 mg / l, and any values ​​and / or subranges therebetween. The duration of the ozone treatment may range from about 10 minutes to about 4 days, and any values ​​and / or subranges therebetween. The ozone treatment may be conducted at a temperature of about 18°C ​​to about 30°C, and any values ​​and / or subranges therebetween. The ozone may be gaseous or liquid. The treatment may include the use of both gaseous and liquid ozone. The ozone treatment may include gaseous ozone and ozone delivered through a liquid medium, for example, nuts may be exposed to gaseous ozone first and then to liquid ozone treatment, or vice versa.

[0078] In certain embodiments, ozone treatment uses gaseous ozone. The dosage of gaseous ozone can be about 120 g / m 3 (grams per cubic meter = mg / l) to 250g / m 3 , about 160g / m 3 Up to 240g / m 3 , about 160g / m 3 , or about 240g / m 3 In one embodiment, the treatment uses about 160 g / m 3 In one embodiment, the treatment uses about 240 g / m 3 of gaseous ozone for 60 minutes.

[0079] In certain embodiments, ozone treatment uses an aqueous ozone solution. The dosage of the aqueous ozone solution may be about 10 g / m 3 (grams per cubic meter = mg / l) to 50g / m 3 , about 20g / m 3 Up to 40g / m 3 , about 20g / m 3 , or about 40g / m 3 In one embodiment, the treatment uses about 20 g / m3 In another embodiment, the treatment is carried out using an ozone solution of about 40 g / m 3 of ozone aqueous solution for 15 minutes.

[0080] In certain embodiments, ozone treatment may include both gaseous ozone and ozone delivered via a liquid medium. For example, nuts may be exposed to gaseous ozone first and then to aqueous ozone treatment, or vice versa. Peroxide treatment may precede, partially or completely overlap with, or follow ozone treatment. In certain embodiments, aqueous ozone treatment may precede peroxide treatment and be followed by gaseous ozone treatment. Peroxide treatment may partially or completely overlap with aqueous ozone treatment.

[0081] Example 4a

[0082] The hazelnuts were collected and their aflatoxin content was determined, for example, by high performance liquid chromatography (HPLC), to be at least about 300 ppb on average. The moisture content of the hazelnuts was determined to be about 3% to 8%. The hazelnuts were in shell. One kilogram of the in-shell hazelnuts was treated with about 1%, about 10%, or about 30% hydrogen peroxide at room temperature for one hour, and then treated with 240 g / cm 3 Nine different treatment combinations were obtained by treating hazelnuts with ozone and hydrogen peroxide for 10, 30, or 60 minutes. For example, high-performance liquid chromatography (HPLC) analysis of aflatoxin levels in hazelnuts following ozone and hydrogen peroxide treatment showed a reduction of at least one log from 300 ppb to, for example, approximately 30 ppb or less.

[0083] Electrolyzed Oxidized Water (EOW)

[0084] In another embodiment of the method, one or more nuts having at least about 300 ppb aflatoxins, and / or the presence of Aspergillus flavus and / or Aspergillus parasiticus at levels resulting in an aflatoxin content of at least about 300 ppb, are contacted with electrolytically oxidized water (EOW) to reduce the aflatoxin content to less than 300 ppb and / or reduce the Aspergillus flavus and / or Aspergillus parasiticus content to a level of less than about 300 ppb aflatoxin. In certain embodiments, the aflatoxin content is reduced by at least half a log (e.g., to about 95 ppb), at least one log (e.g., to about 30 ppb), at least 1.5 logs (e.g., to about 9.5 ppb), or at least two logs (e.g., to about 3 ppb).

[0085] Electrolytically oxidized water (EOW) is produced by electrodialysis of an ionic solution using direct current in an electrolytic cell, where the anode and cathode are separated by a membrane.

[0086] EOW can be acidic electrolytically oxidized water (AEOW), neutral electrolytically oxidized water (NEOW), or alkaline electrolytically oxidized water (BEOW) produced at the cathode. NEOW can be produced by adding hydroxide ions to AEOW or using a single-chamber electrolyzer. AEOW has a pH of 2.5 to 3.5, an oxidation-reduction potential (ORP) of 1000-1200 mV, and an available chlorine content (ACC) of 30 to 90 ppm. NEOW has a pH of approximately 5.0 to 7, an oxidation-reduction potential (ORP) of approximately 700 to 900 mV, and an available chlorine content (ACC) of 30 to 90 ppm. BEOW has a pH of 10 to 13, an oxidation-reduction potential (ORP) of -795 to -900 mV, and an available chlorine content (ACC) of 80 to 100 ppm.

[0087] AEOW and NEOW are preferred embodiments of the present invention. EOW treatment of nuts can be carried out at a temperature of about 20°C to about 50°C, about 25°C to about 45°C, about 22°C to about 27°C, or about 25°C. The ratio of the volume (ml) of the EOW to the mass of the nuts (g) can be at least about 2:1 (v / m), at least about 3:1 (v / m), at least about 4:1 (v / m), or at least about 5:1 (v / m). The treatment time can be from about 5 minutes to about 60 minutes, about 10 minutes to about 30 minutes, about 10 minutes to about 25 minutes, about 10 minutes to about 20 minutes, or about 15 minutes. The treatment can be carried out under static conditions (e.g., the nuts do not move in the EOW) or under moving conditions (e.g., the nuts move in the EOW, such as by shaking). Moving the nuts during processing (e.g. using a mesh conveyor or multi-level conveyor) can accelerate aflatoxin reduction compared to processing under static conditions, especially within the first approximately 15 minutes of processing.

[0088] Example 5a

[0089] Pistachios are collected and their aflatoxin content is determined, for example, by high performance liquid chromatography (HPLC), to an average level of at least about 250 ppb. Hazelnuts are in-shell. One kilogram of in-shell pistachios is subjected to AEOW treatment. The AEOW has a pH of about 2.3, an ORP of about 1100, and an ACC of about 70. The treatment is performed at a 4:1 (v / m) ratio for 15 minutes at a temperature of about 25°C under static conditions. The aflatoxin content of the pistachios after AEOW treatment is determined, for example, by high performance liquid chromatography (HPLC), to be reduced by at least one log from 250 ppb, for example, to about 25 ppb or less.

[0090] Example 5b

[0091] Hazelnuts are collected and their aflatoxin content is determined, for example, by high performance liquid chromatography (HPLC), to an average level of at least about 200 ppb. The hazelnuts are in shell. One kilogram of in-shell hazelnuts is subjected to AEOW treatment. AEOW has a pH of about 2.3, an ORP of about 1100, and an ACC of about 70. The treatment is performed at a ratio of 4:1 (v / m) for 15 minutes under static conditions at a temperature of about 25°C. The aflatoxin content of the hazelnuts after AEOW treatment, for example, by high performance liquid chromatography (HPLC), is determined to be approximately a one-log reduction from 200 ppb, for example, to about 20 ppb or less.

[0092] Pulsed Electric Field (PEF)

[0093] In another embodiment of the method, one or more nuts having at least about 300 ppb aflatoxins, and / or nuts in which Aspergillus flavus and / or Aspergillus parasiticus are present at levels resulting in an aflatoxin content of at least about 300 ppb, are exposed to a pulsed electric field (PEF) to reduce the aflatoxin content to less than 300 ppb and / or reduce the level of Aspergillus flavus and / or Aspergillus parasiticus to a level of less than about 300 ppb aflatoxin. In certain embodiments, the aflatoxin content is reduced by at least half a log (e.g., to about 95 ppb), at least one log (e.g., to about 30 ppb), at least 1.5 logs (e.g., to about 9.5 ppb), or at least two logs (e.g., to about 3 ppb).

[0094] The forces generated by PEF within nuts are sufficient to create and destroy pores in living cells, such as Aspergillus cells. PEF can also lead to the degradation of aflatoxins. In a reaction chamber, the material to be treated is subjected to brief electrical pulses (each lasting from microseconds to milliseconds) at a high voltage (typically 10-100 kV / cm) between electrodes. Process conditions such as electric field strength (kV / cm), pulse frequency, pulse width, pulse waveform, and exposure time (related to the fluid flow and volume in the electrode chamber) can be adjusted as needed to achieve aflatoxin reduction. The product can be treated by the electrodes once or multiple times to achieve aflatoxin degradation. PEF treatment parameters are affected by the size, geometry, and moisture content of the nuts. The electrical pulse energy used in PEF treatment generates heat. In some cases, it is beneficial to incorporate cooling into the process to maintain the treated product at an appropriately low temperature. PEF treatment chambers can be divided into batch chambers for solid material processing, such as batches of nuts.

[0095] Example 6a

[0096] Peanuts are collected and their aflatoxin content is determined, for example, by high performance liquid chromatography (HPLC), to determine an average aflatoxin content of at least about 300 ppb. The peanuts are shelled. A pilot-scale pulsed electric field (PEF) system is used. Two sets of electrodes are positioned within a treatment chamber, allowing the peanuts to freely fall between the parallel electrodes from the top to the bottom of the chamber. The distance between the electrodes is adjusted to ensure that the peanuts do not become blocked as they pass through the treatment chamber. The PEF system can apply an electric field strength of up to 20 kV and delivers monopolar rectangular pulses. The PEF system is designed to allow the peanuts to pass through the electric field one or more times. A pulsed electric field (PEF) with a maximum peak voltage of 10 kV and a frequency of 100 Hz, 140 Hz, 160 Hz, or 180 Hz is applied. The in-shell peanuts are subjected to one to eight cycles of the PEF. The PEF treatment is described by energy level (joules), as the treatment time and frequency are used to calculate the energy applied. The PEF treatment energy ranged from 0.97 joules (J) (for one cycle through the treatment chamber at 100 Hz) to 17.28 J (for eight cycles through the treatment chamber at 180 Hz). The aflatoxin content of the peanuts treated with PEF, as measured by high-performance liquid chromatography (HPLC), was shown to be reduced by at least one log from 300 ppb, for example, to about 30 ppb or less. Treatment conditions were adjusted to achieve a reduction of at least 1.5 logs (for example, to about 9.5 ppb or less).

[0097] Example 6b

[0098] The hazelnuts were collected and their aflatoxin content was determined, for example, by high performance liquid chromatography (HPLC), and the results showed an average content of at least about 200 ppb. The hazelnuts were shelled. A pilot-scale PEF system as described in Example 5a was used. A maximum peak voltage of 10 kV was applied at a frequency of 100 Hz, 140 Hz, 160 Hz, or 180 Hz. The in-shell hazelnuts were subjected to 1 to 8 cycles of PEF treatment. The PEF treatment is described in terms of energy levels (joules) because the treatment time and frequency are used to calculate the energy applied. The PEF treatment range was from 0.97 joules (J) (1 cycle through the treatment chamber at 100 Hz) to 17.28 J (8 cycles through the treatment chamber at 180 Hz). The aflatoxin content of the PEF-treated hazelnuts was determined, for example, by high performance liquid chromatography (HPLC), and the results showed that the content was reduced by at least about 1, for example, from 200 ppb to about 20 ppb or less.

[0099] Supercritical carbon dioxide

[0100] In another embodiment of the method, one or more nuts having at least about 300 ppb aflatoxins, and / or the presence of Aspergillus flavus and / or Aspergillus parasiticus at levels that result in an aflatoxin content of at least about 300 ppb, are contacted with supercritical carbon dioxide (SC CO2) to reduce the aflatoxin content to less than 300 ppb, and / or reduce the content of Aspergillus flavus and / or Aspergillus parasiticus to a level of less than about 300 ppb aflatoxin. In certain embodiments, the aflatoxin content is reduced by at least half a log (e.g., to about 95 ppb), at least one log (e.g., to about 30 ppb), at least 1.5 logs (e.g., to about 9.5 ppb), or at least two logs (e.g., to about 3 ppb).

[0101] Under high pressure, carbon dioxide (CO2) enters a supercritical state, where it possesses properties of both a liquid and a gas. In this supercritical state, CO2 can act as a selective solvent, dissolving specific target compounds by adjusting pressure and temperature. To extract polar molecules, other polar solvents such as ethanol can be added to the CO2 as polarity modifiers to improve the extraction efficiency of these polar target compounds.

[0102] When using supercritical fluid extraction (SFE) for treatment, supercritical carbon dioxide (SC CO2) is used and the pressure can be about 2,000 pounds per square inch (psi) to about 15,000 psi, about 2000 psi to about 10,000 psi, about 2000 psi to about 6000 psi, or about 5000 psi. The temperature can be about 40°C to about 80°C, about 45°C to about 60°C, or about 50°C. The treatment time can be about 5 minutes to about 60 minutes, about 5 minutes to about 30 minutes, about 5 minutes to about 15 minutes, for example, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes or 60 minutes. SC CO2 has a polar co-solvent. Typical polar co-solvents are ethanol, isopropanol or water. In certain embodiments, the content of the polar co-solvent can be about 5% to about 20%. In certain embodiments, the ratio of polar solvent volume (ml) to nut weight (g) can be 1 to 4, such as 1.5, 2, 3, or 4.

[0103] Example 7a

[0104] Cashew nuts are collected and their aflatoxin content is measured, for example, by high performance liquid chromatography (HPLC), to determine an average aflatoxin content of at least about 300 ppb. The cashew nuts are shelled. One kilogram of shelled cashew nuts is placed in an extraction chamber with 10% ethanol (at a ratio of 3 ml / g ethanol to cashew nuts) and pressurized to 5000 psi with CO2. The treatment is conducted at 50°C for 15 minutes. The aflatoxin content of the cashew nuts after the SC CO2 treatment is measured, for example, by high performance liquid chromatography (HPLC), and the results show a reduction of at least one log from 300 ppb (e.g., to about 30 ppb or less).

[0105] Example 7b

[0106] The hazelnuts are collected and their aflatoxin content is determined, for example, by high-performance liquid chromatography (HPLC), to an average value of at least about 200 ppb. The hazelnuts are shelled. One kilogram of shelled hazelnuts is placed in an extraction chamber with 10% ethanol (3 ml / g ethanol to hazelnuts) and pressurized to 5000 psi with CO₂. The treatment is carried out at 50°C for 15 minutes. The aflatoxin content of the hazelnuts after the SC CO₂ treatment is determined, for example, by high-performance liquid chromatography (HPLC), to show a reduction of at least one log from 200 ppb, for example, to about 20 ppb or less.

[0107] cold plasma

[0108] In another embodiment of the method, one or more nuts having at least about 300 ppb aflatoxins, and / or the presence of Aspergillus flavus and / or Aspergillus parasiticus in an amount resulting in an aflatoxin content of at least about 300 ppb, are contacted with a cold plasma to reduce the aflatoxin content to less than 300 ppb and / or reduce the level of Aspergillus flavus and / or Aspergillus parasiticus to a level of less than about 300 ppb aflatoxin. In certain embodiments, the aflatoxin content is reduced by at least half a log (e.g., to about 95 ppb), at least one log (e.g., to about 30 ppb), at least 1.5 logs (e.g., to about 9.5 ppb), or at least two logs (e.g., to about 3 ppb).

[0109] In practicing the methods of the present invention, exemplary systems for generating cold atmospheric pressure plasma (CAP or CAPP) (but not limited to) include dielectric barrier discharge (DBD) plasma systems and air surface barrier discharge (SBD) systems. Other systems, such as high-pressure atmospheric cold plasma systems, atmospheric pressure fluidized bed plasma systems, and low-pressure cold plasma systems, may also be used. Low-pressure, high-humidity plasma is another exemplary plasma. Exemplary gases include nitrogen (N2) and nitrogen-containing mixtures, such as N2 with 0.1% to 1% O2, such as N2 + 0.1% O2 and N2 + 1% O2, and ambient air. Optionally, the relative humidity (RH) of the gas is between 5% and 80%, such as 20% RH, 40% RH, and 80% RH. For SBD systems employing this method, the power can be between about 0.18 W / cm3 and 0.31 W / cm3, and the exposure duration can be between about 1 minute and about 8 minutes, such as 1 minute, 2 minutes, 4 minutes, or 8 minutes. For a DBD system employing this method, the power can range from about 400 W to about 1150 W, and the exposure duration can be from about 2 minutes to about 12 minutes, such as 2 minutes, 4 minutes, 8 minutes, 10 minutes, or 12 minutes. The exposure duration is inversely proportional to the power. Higher powers may result in elevated temperatures, which may not be desirable. The elevated temperature can be mitigated, for example, by using lower powers and longer exposure durations.

[0110] Example 8a

[0111] The walnuts are collected and their aflatoxin content is measured, for example, by high performance liquid chromatography (HPLC), to determine an average level of at least about 250 ppb. The moisture content of the walnuts is measured to determine a moisture content of about 3% to 8%. The walnuts are in-shell. One kilogram of the in-shell walnuts is treated with a cold atmospheric pressure plasma from a DBD system using N2 + 0.1% O2 at 700 W for 4 minutes. The aflatoxin content of the walnuts treated with the cold atmospheric pressure plasma is measured, for example, by high performance liquid chromatography (HPLC), to determine a reduction of at least one log from 250 ppb (e.g., to about 25 ppb or less).

[0112] Example 8b

[0113] The hazelnuts are collected and their aflatoxin content is determined, for example, by high performance liquid chromatography (HPLC), to be an average of at least about 200 ppb. The moisture content of the hazelnuts is measured and determined to be about 3% to 8%. The hazelnuts are in-shell. One kilogram of the in-shell hazelnuts is treated with cold atmospheric pressure plasma from a DBD system using N2 + 0.1% O2 at 700 W for 4 minutes. The aflatoxin content of the hazelnuts treated with cold atmospheric pressure plasma is determined, for example, by high performance liquid chromatography (HPLC), and the results show that the aflatoxin content is reduced by at least one log from 200 ppb (e.g., to about 20 ppb or less).

[0114] Organic acid washing

[0115] In another embodiment of the method, one or more nuts having at least about 300 ppb aflatoxins, and / or the presence of Aspergillus flavus and / or Aspergillus parasiticus at levels resulting in an aflatoxin content of at least about 300 ppb, are contacted with an organic acid solution to reduce the aflatoxin content to less than 300 ppb and / or reduce the level of Aspergillus flavus and / or Aspergillus parasiticus to a level of less than about 300 ppb aflatoxin. In certain embodiments, the aflatoxin content is reduced by at least half a log (e.g., to about 95 ppb), at least one log (e.g., to about 30 ppb), at least 1.5 logs (e.g., to about 9.5 ppb), or at least two logs (e.g., to about 3 ppb).

[0116] Organic acids are organic molecules containing acidic groups. They are typically weak acids and therefore milder than strong acids such as hydrochloric acid and sulfuric acid. Typical organic acids that can be used in the methods of the present invention include citric acid, lactic acid, propionic acid, acetic acid, and tartaric acid. In certain embodiments, the organic acid is citric acid, lactic acid, or propionic acid. The concentration of the organic acid solution can be between 1% (w / w) and 10% (w / w), for example, 1%, 3%, 5%, 7%, or 9%. In certain embodiments, the concentration of the organic acid solution is between 3% and 7%. The moisture content of the nuts can affect the effectiveness of the chemical cleaning agent. Nuts with a higher moisture content (e.g., 16%) are more suitable for using higher concentrations of organic acid than nuts with a lower moisture content (e.g., 10%). The treatment can be carried out at room temperature, for example, between about 20°C and about 30°C, such as 20°C, 22°C, 25°C, or 27°C. The treatment time can be between 5 minutes and 30 minutes, for example, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, or 30 minutes. The ratio of nuts to organic acid solution (ml) to nuts (g) can be 0.5 ml / g to 5 ml / g, for example 0.5 ml / g, 1 ml / g, 2.5 ml / g, or 5 ml / g. In certain embodiments, the ratio of nuts to organic acid solution (ml) to nuts (g) can be 1 ml / g. The treatment can be performed under static conditions (e.g., the nuts are not moved in the organic acid solution) or under moving conditions (e.g., the nuts are moved in the organic acid solution, such as by shaking).

[0117] Example 9a

[0118] Peanuts are collected and their aflatoxin content is measured, for example, by high performance liquid chromatography (HPLC), to determine an average aflatoxin content of at least about 300 ppb. The moisture content of the peanuts is measured and determined to be about 10%. The peanuts are in their shells. One kilogram of in-shell peanuts is treated with a 3% citric acid solution (approximately 1 ml of citric acid solution per gram of peanuts) at 27°C for 15 minutes. The aflatoxin content of the peanuts after organic acid treatment is measured, for example, by high performance liquid chromatography (HPLC), to determine a reduction of at least one log from 300 ppb, for example, to about 30 ppb or less. Treatment conditions (e.g., concentration, treatment time, temperature, etc.) are adjusted to reduce the aflatoxin content by about two logs.

[0119] Example 9b

[0120] The hazelnuts are collected and their aflatoxin content is determined, for example, by high-performance liquid chromatography (HPLC), to an average level of at least about 200 ppb. The moisture content of the hazelnuts is determined to be approximately 10%. The hazelnuts are in-shell. One kilogram of the in-shell hazelnuts is treated with a 3% citric acid solution (approximately 1 ml of citric acid solution per gram of hazelnuts) at 27°C for 15 minutes. The aflatoxin content of the hazelnuts after organic acid treatment is measured, for example, by HPLC, and the results show a level of less than 200 ppb.

[0121] Combined processing

[0122] In another aspect, the nuts may be treated with two different technologies, or more than two different technologies. The nuts may be treated with the two different technologies substantially simultaneously (i.e., in parallel), or sequentially, or the different technologies may overlap during treatment, e.g., a portion of the UV treatment may be performed alone, while another portion may be overlapped with ozone and / or hydrogen peroxide. For example, the nuts may receive a first treatment and a second treatment simultaneously. Alternatively, the nuts may receive a first treatment and a second treatment consecutively. Furthermore, one or both of the two technologies may be repeated, and / or additional technologies may be used. It is contemplated that a combination of treatments may be used to achieve a reduction in the dose, intensity, and / or duration of a single treatment regimen while achieving an overall reduction in aflatoxins, and / or Aspergillus flavus and / or Aspergillus parasiticus, which cause aflatoxin contamination. It is also contemplated that a combination of treatments can further reduce aflatoxins and / or reduce the levels of Aspergillus flavus and / or Aspergillus parasiticus, which cause aflatoxin contamination. In certain embodiments, after at least two treatments, the aflatoxins are reduced by at least half a log, at least a log, at least 1.5 logs, at least 2 logs, or at least 2.5 logs.

[0123] In one embodiment of the present invention, nuts are treated with ultraviolet (UV) light and a peroxide (e.g., hydrogen peroxide). In one embodiment, the UV light treatment and the hydrogen peroxide treatment are performed sequentially. In one embodiment, the UV light treatment and the hydrogen peroxide treatment are performed nearly simultaneously. It is expected that UV light can accelerate the generation of free radicals from hydrogen peroxide, thereby enhancing the reduction of aflatoxins. Treatment conditions can be adjusted as needed. For example, the amount of hydrogen peroxide used can be 0.1% H2O2 to 10% H2O2.

[0124] In one embodiment of the present invention, the nuts are subjected to ultraviolet (UV) irradiation and ozone treatment. In one embodiment, the nuts are subjected to UV irradiation and ozone treatment sequentially. In one embodiment, the nuts are subjected to UV irradiation and ozone treatment almost simultaneously. In one embodiment, the UV treatment may partially overlap with one or more ozone treatments. In one embodiment, the UV light is from about 350 nm to about 365 nm, or from about 140 nm to about 350 nm. In one embodiment, the UV light is from about 200 nm to 280 nm, for example, 254 nm or 256 nm. In another embodiment, the UV light wavelength is less than about 200 nm, for example, about 186 nm or about 142 nm.

[0125] In one embodiment of the present invention, nuts are treated with ozone and peroxide. In one embodiment, the ozone treatment and the peroxide treatment are applied to the nuts sequentially. In one embodiment, the ozone treatment and the peroxide treatment are applied to the nuts approximately simultaneously. In one embodiment, the peroxide treatment may overlap with one or more ozone treatments. The ozone treatment may be gaseous ozone, aqueous ozone, or a combination of both.

[0126] In one embodiment of the present invention, the nuts are first irradiated with ultraviolet (UV) light and then treated with supercritical carbon dioxide.

[0127] In one embodiment of the present invention, nuts are first treated with a pulsed electric field (PEF) and then with peroxide.

[0128] In one embodiment of the present invention, the nuts are first treated with peroxide and then with supercritical carbon dioxide.

[0129] Further combinations of treatments can be employed when practicing the disclosed methods. Furthermore, combinations of three or more treatments are contemplated when practicing the methods of the present invention. Nuts can be treated using three or more different techniques simultaneously (i.e., in parallel), or they can be treated sequentially using three or more treatments, or a combination of both. For example, nuts can be treated simultaneously with a first treatment and a second treatment, followed by a third treatment. Furthermore, one or more of the three different techniques can be repeated.

[0130] In one embodiment of the present invention, the nuts are treated with ultraviolet (UV) light, ozone and peroxide.

[0131] In one embodiment of the present invention, the nuts are treated with ultraviolet (UV) light, ozone and citric acid.

[0132] Artificially contaminated nuts

[0133] The present invention also relates to a method for preparing nuts contaminated with aflatoxin and / or Aspergillus. This method provides nuts with controlled contamination levels and reliable sources, and supports in-situ production. Contaminated nuts can be used to test, develop, and / or optimize decontamination methods in a more controlled manner than naturally contaminated nuts.

[0134] Example 10: Nuts contaminated with Aspergillus

[0135] To develop a method for preparing aflatoxin-contaminated nuts, three Aspergillus strains were identified with a target aflatoxin contamination level of 200 ppb. All three strains produced aflatoxins B1 and B2. The A. parasiticus strain also produced aflatoxins G1 and G2.

[0136] Each strain was tested for growth and aflatoxin production on clean nuts using two sets of culture conditions and six time points. The test temperatures were 25°C and 30°C, and the time points covered 0-10 days. The humidity was fixed at >97% relative humidity (RH). The nuts used were hazelnuts. The experimental process included: (1) preparing Aspergillus; (2) preparing hazelnuts; (3) inoculating hazelnuts; and (4) cultivating aflatoxin. These steps are described in detail below.

[0137] Preparation of Aspergillus

[0138] 1. Prepare 10 x 90 mm malt extract agar (MEA) plates and inoculate Aspergillus flavus (ATCC #11498; CABI #52140), Aspergillus parasiticus (ATCC #16875; CABI #124931), and Aspergillus flavus (ATCC #22546; CABI #370082). Although CABI #124931 is labeled as Aspergillus parasiticus in its catalog, ATCC #16875 is labeled as Aspergillus flavus. The Aspergillus flavus strain ATCC 16875 is cataloged in various strain collections as: CBS 573.65; IFO 7540; QM 6738; WB 500; and NRRL 500.

[0139] 2. Incubate the plates at 25°C for 10 days.

[0140] 3. Confirm spore formation by microscopic observation.

[0141] 4. In a biosafety cabinet, collect spores by swabbing and resuspend in 20 ml sterile deionized water (SDWT) containing 0.1% Tween.

[0142] 5. Transfer the spore suspension to a centrifuge tube and pellet the spores by centrifugation (4,000 rpm, 10 minutes).

[0143] 6. Discard the supernatant and resuspend the spore pellet in 20 mL of SDWT.

[0144] 7. Spore counts were estimated using a Neubauer counting chamber (WI-MB-11-025) and confirmed by enumerating spore suspensions on Aspergillus flavus and Aspergillus parasiticus agar (AFPA) in triplicate.

[0145] 8. Adjust the concentration to 10 5 CFU / mL.

[0146] 9. Store the suspension at 2-8°C until use (up to 1 week).

[0147] Preparing the nuts

[0148] 1. Determine the initial water activity and moisture content of hazelnuts.

[0149] 2. Soak the samples in 1% sodium hypochlorite solution for 10 minutes to reduce the content of natural microorganisms / fungi.

[0150] 3. Rinse with sterile distilled water to remove residual sodium hypochlorite.

[0151] 4. Use absorbent paper to remove most of the remaining water.

[0152] 5. For damaged hazelnuts, first put 1100 grams of hazelnuts into the cement mixer and run it for 3 hours.

[0153] Inoculated nuts

[0154] 1. Place three 1,100g hazelnut samples into large sterile mixing bags.

[0155] 2. Add approximately 15 ml of A. parasiticus or A. flavus spore suspension to each bag and mix thoroughly by hand to ensure that the inoculum is evenly distributed in the sample.

[0156] 3. Transfer the sample to a tray covered with filter paper and dry at room temperature under laminar filtered air flow.

[0157] 4. Check water activity regularly until it returns to the pre-vaccination level.

[0158] 5. Analyze three replicate samples to determine the initial inoculum level achieved after drying.

[0159] Aflatoxin culture

[0160] 1. Transfer the sample to a sealed 50L box containing 15L of sterile deionized water. Place the nut sample in a single layer on a mesh tray and suspend it above the water at a relative humidity of >97%. Use a calibrated hygrometer to confirm humidity.

[0161] 2. Incubate the samples at 30±2°C for 4 days.

[0162] 3. After the incubation period, take a small portion of each batch and image it under UV light. Use uninoculated hazelnuts as a control image.

[0163] 4. Nut samples were taken immediately after incubation, washed (both conditions) and measured for aflatoxin levels to determine tolerance to contamination.

[0164] Washing condition 1: water, 10 minutes

[0165] Wash condition 2: 1% hypochlorite, 10 minutes, followed by water rinse

[0166] 5. Spread the nuts on trays and dry until their water activity / moisture content returns to pre-inoculation levels.

[0167] 6. Take five 50g samples from each batch and use r- A total aflatoxin ELISA kit (R-Biopharm AG, Darmstadt, Germany) was used in combination with an aflatoxin immunoaffinity column to determine the aflatoxin content.

[0168] 7. Take three replicate samples from each batch on the 7th, 14th and 21st days respectively and use r- A total aflatoxin ELISA kit (R-Biopharm AG, Darmstadt, Germany) was used to determine the aflatoxin content.

[0169] Significant mold growth was observed on the hazelnuts for all three strains and at both temperatures tested. Mold growth was significantly more extensive in the cultured samples compared to native hazelnuts, which is attributed to the accelerated growth period. Mold culture results showed that the mold on the nuts was exclusively Aspergillus spp., attributed to the use of a sodium hypochlorite solution to wash the nuts prior to inoculation; no signs of other mold species were observed.

[0170] Data such as Figure 3As shown in Figure 3 . Significant variability in aflatoxin levels was observed. While not strictly theoretical, this significant variability is believed to be due to two main factors. First, the degree of contamination varied significantly between nuts due to varying degrees of shell damage and corresponding levels of mold growth. Although this variability was large, the level of variability observed in this study (approximately one order of magnitude) was much lower than that observed in naturally grown nuts (3-4 orders of magnitude). Second, the sample size for these measurements was small (10 grams, approximately 11 nuts). Technical screening tests on larger batches of nuts are expected to reduce this uncertainty.

[0171] Results from the Aspergillus contamination experiments showed that several viable combinations of strains and culture parameters could be used to produce samples with a target contamination level of 200 ppb. Aspergillus flavus (ATCC#16875; CABI#124931) was chosen due to its stable growth characteristics and tolerance to small fluctuations in incubation time and temperature while still achieving the desired contamination level. The final contamination level was 216 ± 75 ppb aflatoxin (30°C, day 4), consistent with the target value of 200 ppb.

[0172] Example 11: Aflatoxin-contaminated nuts

[0173] Nuts were mixed with aflatoxin solutions of varying volumes and concentrations. Aflatoxin B1 solutions were prepared in methanol due to the high volatility and solubility of aflatoxins. Aflatoxin content and coating efficiency were determined.

[0174] The experimental plan is as follows:

[0175] 1. Dissolve 5 mg of aflatoxin B1 standard in 20 mL of methanol (250 mg / L) to prepare aflatoxin B1 stock solution.

[0176] 2. Transfer 1,336 μL (equivalent to 334 μg of aflatoxin B1) to a separate 20 mL brown volumetric flask. Fill to the mark with methanol and mix thoroughly by inverting the flask. This concentration is 200 μg / kg (ppb), which corrects for a retention rate of approximately 60% for hazelnuts.

[0177] 3. In a Class I safety cabinet, carefully add the prepared solution to a sterile bag containing 1 kg of hazelnuts to achieve a contamination level of 334 μg / kg (ppb) aflatoxin B1, ensuring that the solution does not come into contact with the upper part of the bag.

[0178] 4. Carefully heat seal the bag to ensure a secure seal.

[0179] 5. Mix thoroughly by hand for 2-3 minutes to evenly distribute the toxin on the surface of the hazelnuts.

[0180] 6. Leave the sample in the safety cabinet and wait for the aerosol to settle completely.

[0181] 7. In a safety cabinet, pour the contaminated hazelnuts onto a tray lined with filter paper to obtain a uniform single layer.

[0182] 8. Dry overnight at room temperature in a Class I safety cabinet with continuous ventilation to allow the methanol to evaporate completely, leaving aflatoxin on the surface of the nuts.

[0183] 9. Transfer the entire batch of samples to a large plastic bag and mix gently but thoroughly.

[0184] 10. Ten 75 g samples were randomly selected from each batch and evaluated for contamination success rate, contamination degree and homogeneity by the selected method (ELISA for Aspergillus inoculated samples and HPLC for direct aflatoxin contamination samples).

[0185] The data are shown in Table 2. The overall contamination level was 216 ± 25 ppb (sample size 30 g), consistent with the target value of 200 ppb. The coating efficiency was approximately 60%, and it was clear that some aflatoxin remained inside the mixing bag and drying tray.

[0186] Table 2

[0187]

[0188]

[0189] The present invention is further described in detail by the above-mentioned experimental examples. These examples are for illustrative purposes only and, unless otherwise indicated, should not be construed as limiting the present invention. Therefore, the present invention should in no way be construed as being limited to the following examples, but should be construed to encompass any and all variations that become apparent from the teachings provided herein.

[0190] The entire contents of each patent, patent application and publication cited in this specification are hereby incorporated into this specification in their entirety by reference.

[0191] Although the embodiments of the present invention have been described in detail through the above examples, it will be understood by those skilled in the art that various modifications may be made without departing from the spirit of these embodiments, and such modifications will be apparent to those skilled in the art. The appended claims are intended to cover all such embodiments and equivalent variations.

Claims

1. A method of reducing aflatoxin contamination in one or more nuts, and / or reducing the level of Aspergillus flavus and / or Aspergillus parasiticus that cause aflatoxin contamination in one or more nuts, the method comprising contacting the one or more nuts with one or more treatments, thereby reducing the presence of aflatoxins and / or reducing the level of Aspergillus flavus and / or Aspergillus parasiticus that cause the presence of aflatoxins, compared to the one or more nuts before contact with the one or more treatments.

2. The method according to claim 1, wherein The one or more treatment methods are selected from the group consisting of ultraviolet (UV) rays, ozone (O3), peroxide, electrolytic oxidized water (EOW), pulsed electric field (PEF), supercritical carbon dioxide, cold plasma, organic acid washing, or a combination thereof.

3. The method according to claim 1 or claim 2, wherein: The one or more nuts are contacted with two treatment methods selected from the group consisting of ultraviolet (UV) light, ozone (O3), peroxide, electrolytic oxidized water (EOW), pulsed electric field (PEF), supercritical carbon dioxide, cold plasma, organic acid washing, or a combination thereof.

4. The method according to claim 2, wherein The one or more nuts are contacted with two treatments selected from the group consisting of ultraviolet (UV) light, ozone (O3), and peroxide.

5. The method according to claim 2, wherein The one or more nuts are contacted with ultraviolet (UV) light and ozone (O3), or with ultraviolet (UV) light and peroxide.

6. The method according to claim 2, wherein The one or more nuts are contacted with ozone (O3) and a peroxide.

7. The method according to claim 2, wherein The one or more nuts are contacted with ultraviolet (UV) light, ozone (O3) and peroxide.

8. The method according to claim 1 or claim 2, wherein: The one or more nuts are contacted with three treatment methods selected from the group consisting of ultraviolet (UV) light, electrolytically oxidized water (EOW), ozone (O3), peroxide, pulsed electric field (PEF), supercritical carbon dioxide, cold plasma, organic acid washing, or a combination thereof.

9. The method according to claim 2, wherein The one or more nuts are contacted with ultraviolet (UV) light, ozone (O3) and peroxide.

10. The method according to claim 1, wherein The nuts are almonds, cashews, hazelnuts, macadamia nuts, peanuts, pecans, pistachios, walnuts, or combinations thereof.

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