REDUCTION OF DEOXYNIVALENOL IN CEREAL GRAINS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to United States Provisional Application 63/413,111, filed on October 4, 2022, the entire contents of which are incorporated herein by reference, where permitted.

FIELD OF THE INVENTION

[0002] The invention relates to methods and systems for reducing deoxynivalenol in cereal grains during malt production, including for malting barley, and improving the germination rate of cereal grains.

BACKGROUND OF THE INVENTION

[0003] Barley is the most widely used cereal grain for malt production. Barley grains can be contaminated with fungal pathogens that produce certain mycotoxins in the field or during storage. One of the most common mycotoxins in malt and beer is deoxynivalenol (DON), which is mainly produced by Fusarium graminearum. DON has various toxic effects in humans and animals, causing acute temporary diarrhea, nausea, vomiting, headache, dizziness, fever, and abdominal pain. In addition, contamination of grains by DON can lead to a significant crop yield loss. There are strict guidelines regarding the presence of mycotoxins in food and feed. Agriculture and Agri -Food Canada has set 1 pg/g as the acceptable level of DON for swine, and dairy cattle, and 5 pg/g for beef cattle, and poultry [1],

[0004] The common postharvest methods for removing mycotoxins from food and feed are physical, chemical, enzymatic and microbial methods. Malt and grain industries continue to seek for better technologies to degrade mycotoxins in food and feed, since the current methods are time consuming, laborious, expensive, energy intensive, or may negatively impact the quality of the treated product. SUMMARY OF THE INVENTION

[0005] In general terms, this disclosure provides the use of plasma activated water (PAW) as a substitute for water during the steeping process in order to reduce deoxy nival enol (DON) concentration in a cereal grain, such as in naturally infected barley (NIB). This use of PAW also has a beneficial effect on the germination and quality parameters of barley malt. Development of the invention included a determination of the major degradation products of DON after ACP treatment.

[0006] In one aspect, this disclosure provides a method of steeping barley in a malting process, comprising the step of steeping the barley in plasma-activated water (PAW). In some embodiments, the barley is treated by immersing in water while generating atmospheric cold plasma in the water to produce PAW. This active ACP treatment may continue through steeping, or may be discontinued and followed by steeping. In some embodiments, the PAW is generated first and then used to steep the barley.

[0007] In some embodiments, the barley is steeped in PAW for at least 30 min, such as between about 30 minutes and 5 hours. The barley may then be allowed to rest in air for an additional period of time, preferably at least about 1 hour, such as between about 1 hour and 24 hours.

[0008] In some embodiments, the PAW may be recirculated from a steeping tank to be treated again with ACP prior to be reintroduced to the steeping tank.

[0009] In another aspect, this disclosure provides a system for supplying plasma-activated water to a steeping tank, comprising:

(a) a water source connected to the steeping tank; and

(b) a plasma generator having a plasma outlet in the water source.

[0010] In some embodiments, the steeping tank has a recirculation outlet, for flowing water back to the water source, or to combine with the water source. [0011] In some embodiments, the plasma outlet discharges into a water conduit, which preferably comprises a venturi leading to the steeping tank.

[0012] In another aspect, this disclosure provides a system for steeping barley in a malting process, comprising:

(a) a steeping tank for steeping the barley; and

(b) a plasma generator comprising a plasma diffuser disposed in the steeping tank, an air inlet, a ground electrode and a high voltage electrode.

[0013] In accordance with at least one broad aspect, there is provided a system for steeping cereal grains in a malting process, comprising:

(a) a steeping tank for steeping the cereal grain, and receiving a volume of water; and

(b) at least one plasma generator to produce plasma activated water (PAW) inside the steeping tank.

[0014] In some embodiments, the cereal grains are naturally infected barley (NIB) grains.

[0015] In some embodiments, the at least one plasma generator includes an outlet, which is inserted into water located in the steeping tank.

[0016] In some embodiments, the system further comprises a water source coupled to the steeping tank, via a water conduit.

[0017] In some embodiments, the at least one plasma generator includes an outlet which is inserted into the water source.

[0018] In some embodiments, the at least one plasma generator includes an outlet which is coupled to the water conduit.

[0019] In some embodiments, the water conduit comprises a design configured to promote the venturi effect. [0020] In some embodiments, the system further comprises a recirculation conduit, extending between a recirculation outlet of the steeping tank, and the water source.

[0021] In some embodiments, the plasma generator comprises a plasma diffuser, a gas inlet, a ground electrode and a high voltage electrode.

[0022] In some embodiments, the gas source is coupled to the gas inlet, and the gas source comprises pressurized air.

[0023] In another broad aspect, there is provided a system for steeping cereal grains using plasma activated water (PAW), comprising:

(a) a water source;

(b) a gas source;

(c) a PAW production tank connected to the water source and having an outlet connected to a steeping tank for steeping the cereal grain; and

(d) a plasma generator connected to the gas source, and having an outlet for feeding plasma into the PAW production tank.

[0024] In another broad aspect, there is provide a method of steeping barley in a malting process, comprising: operating a plasma generator, to generate atmospheric cold plasma (ACP); treating water with ACP to produce plasma activated water (PAW); and steeping cereal grain in the PAW.

[0025] In some embodiments, the cereal grains are naturally infected barley (NIB) grains.

[0026] In some embodiments, the barley is steeped for between 1 hour and 5 hours, preferably 4.5 h, after ACP generation. [0027] In some embodiments, the cereal grain is subject to air rest following steeping.

[0028] In some embodiments, the cereal grain is subject to air rest for 1 hour and 24 hours.

[0029] In some embodiments, treating and steeping occur concurrently or partially concurrently.

[0030] In some embodiments, treating occurs prior to the steeping.

[0031] In some embodiments, the method further comprises feeding gas into the plasma generator, to generate the ACP.

[0032] In some embodiments, the gas comprises air or nitrogen.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In the drawings, like elements may be assigned like reference numerals. The drawings are not necessarily to scale, with the emphasis instead placed upon the principles of the present invention. Additionally, each of the embodiments depicted are but one of a number of possible arrangements utilizing the fundamental concepts of the present invention.

[0034] FIG. 1 is a schematic diagram of PAW generation and treatment of cereal grains, where ACP is introduced into a steeping tank.

[0035] FIG. 2 is a schematic diagram of a system for continuously producing PAW for supply to a steeping tank, with optional recirculation.

[0036] FIG. 3 is an example method for PAW generation and treatment of cereal grains.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

I. ABBREVIATIONS.

[0037] The following abbreviations may be used throughout the specification:

ACN (acetonitrile) ACP (atmospheric cold plasma)

DBD (dielectric barrier discharge)

DBE (double-bond equivalent)

DON (deoxynivalenol)

DW (distilled water)

HPLC (high-performance liquid chromatography)

NIB (naturally infected barley)

PAW (plasma activated water)

RNS (reactive nitrogen species)

ROS (reactive oxygen species)

II. GENERAL OVERVIEW.

[0038] It has been appreciated that atmospheric cold plasma (ACP) is capable of degrading deoxynivalenol (DON). Plasma is the fourth state of matter, comprising of reactive species including atoms, and molecules in the ground or excited states, ions, free radicals, electrons, and ultraviolet radiation. In air plasma discharge, a variety of long-lived and short-lived reactive oxygen species are produced, including hydroxyl radical ( OH), singlet oxygen (1O ² ), hydrogen peroxide (H2O2), superoxide anions (O2 '), and ozone (O3) and reactive nitrogen species such as nitrites (NO2 ), nitrate (NO3 ), peroxynitrite (ONOO-), and nitrous oxides (NOx), along with high-energy electrons and ultraviolet light.

[0039] Plasma can be produced by different gas discharge sources including corona discharge, glow discharge, plasma jet, and dielectric barrier discharge. ACP can be used to activate water to produce plasma activated water (PAW). The direct or indirect interaction of reactive species with water molecules, including reactive oxygen species (ROS) and reactive nitrogen species (RNS) in plasma, can change the oxidation-reduction potential and pH of water [0040] In view of the foregoing, disclosed is the use of PAW as a substitute for water during the steeping process in order to reduce the DON concentration in a cereal grain, such as in naturally infected barley (NIB).

[0041] To that end, systems and methods of reducing DON in malting barley using plasma activated water (PAW) are described. Some embodiments comprise the use of PAW as a substitute for water during barley steeping in malting process. Use of PAW for steeping, in one example treatment, resulted in 58.4% reduction in DON concentration.

[0042] Exemplary bubble reactor systems for producing PAW in a steeping tank or supplying PAW to a steeping tank are exemplified schematically in FIGs. 1 and 2.

[0043] In some disclosed embodiments, the PAW comprises the reactive oxygen species ozone and hydrogen peroxide, and the system demonstrated an increase in ORP and a decrease in pH compared to distilled water (DW), which are believed to play a role in DON degradation.

[0044] In at least one example, the a-amylase and P-glucanase activity values of barley grains steeped in PAW were not significantly different compared to distilled water, while P- amylase activity increased compared to control. These findings suggest that the malting industry may use PAW for steeping in order to improve grain germination as well to degrade DON. A study of putative DON degradation products after plasma treatment also indicated the formation of several number of possible DON degradation products (results not shown).

[0045] Without restriction to a theory, the possible structure of major degradation products of DON suggests that oxidation could be the main degradation mechanism of DON by plasma treatment. However, the present disclosure does not rely on any underlying DON degradation mechanism(s), only the fact of degradation. III. EXAMPLE SYSTEMS.

[0046] The following is a description of various example system configurations for PAW generation and treatment of cereal grains (e.g., NIB grains).

[0047] FIG. 1 is a schematic diagram of an example system (100) for steeping cereal grains (e.g., NIB grains), using plasma activated water (PAW).

[0048] As shown, system (100) includes a steeping tank (102) (also referred to herein as a steeping container, or a steeping receptacle). Cereal grains (104) can be placed (i.e., disposed) inside the steeping tank (102), during a steeping process. For example, NIB grains can be placed inside the steeping tank (102) and steeped, as part of a malting process.

[0049] A plasma gas reactor unit (108) (also referred to herein as a plasma generating unit, or a plasma generator), is operated to generate atmospheric cold plasma (ACP).

[0050] The plasma unit (108) can include at least one outlet end (108a), which feeds into water disposed inside the steeping tank (102). For example, outlet (108a) can extend through an open end (102') of the steeping tank (102). In other examples, the outlet (108a) may be disposed in any other position, as desired. Outlet (108a) feeds ACP into the water, to generate plasma bubbles (150), and in turn, produce plasma activated water (PAW) (106).

[0051] In some examples, the cereal grains (104) are immersed into the water, as the PAW (106) is being produced (i.e., as the water is treated with ACP). That is, the cereal grains (104) are immersed into water, while the plasma reactor unit (108) is activated. In other examples, the water inside tank (102) is initially treated with ACP to produce PAW (106), and subsequently, the cereal grains (104) are immersed into the PAW (106).

[0052] As further exemplified, to enable operation of the plasma reactor unit (108), the reactor unit (108) may be coupled to a power supply (112) and a gas tank (116). Power supply (112) provides power via power cable (114), to power the plasma reactor unit (108). Gas tank (116) can contain pressurized air, and may also be coupled to the reactor unit (108), e.g., via the gas supply line (116). The pressurized air can be used to generate the plasma bubbles (150), to produce PAW (106). In other examples, the gas tank (116) can be provided with any other gas suitable for producing an ACP jet (e.g., nitrogen).

[0053] FIG. 2 is a schematic diagram of an example system (200) for steeping cereal grains (e.g., NIB grains), using plasma activated water (PAW).

[0054] System (200) is generally similar to system (100), but allows for continuous production of PAW for supply to the steeping tank (102), with optional recirculation.

[0055] As shown, the system (200) includes a water source (206). Water source (206) can provide a source, or feed (e.g., stream) of water, that can be treated to form plasma activated water (PAW).

[0056] In this example, the water source (206) is fluidically coupled to the steeping tank (102), which retains the cereal grains (104). For example, a water conduit (216) may convey water (210) from the water source (206) to the steeping tank (102). The water source (206) can be, for example, a water tank or any other source of water. In some cases, a pump (208) is provided to facilitate transfer of water through the water conduit (216).

[0057] The outlet (108a), of the plasma reactor unit (108), can feed (directly or in-directly) into the water conduit (216) (e.g., at coupling point (212)). In this manner, the outlet (108a) can feed an ACP jet into the water stream (210), as it is carried to the steeping tank (102). As such, steeping tank (102) can receive a ready supply of PAW (106). To that end, the steeping tank (102) can include a tank inlet (102a), for receiving the flow of PAW (106).

[0058] In at least one example, the water conduit (216) - or a portion thereof - is designed to promote the venturi effect. For example, at least a portion of the water conduit (216) may be designed with a venturi tube. The venturi tube can be constricted in cross-sectional area along a portion of its length, before expanding in cross-sectional area (e.g., in the direction of water flow between water source (206) and steeping tank (102)). In this configuration, the coupling point (212) can occur along the conduit's length, prior to the expanding cross- sectional area of the water conduit (216). [0059] More generally, in accordance with the continuity equation (as known in the art), the velocity of the liquid through a venturi tube increases due to the changing cross-sectional area (i.e., resulting in a pressure difference). The increased velocity allows for the continuous production of plasma bubbles, and also results in turbulence that enhances mixing of the plasma reactive oxygen and nitrogen species with water flowing through the venturi tube. Additionally, when water flow leaves the venturi tube, the formed bubbles implode due to the pressure differential, which also leads to better mixing of the reactive species with water.

[0060] As exemplified, the system (200) can optionally allow for recirculation of water for producing PAW. For example, one or more recirculation conduits (218) may fluidically couple between the steeping tank (102) and the water source (206). For instance, the steeping tank (102) can include a tank outlet (102b) (e.g., a recirculation outlet (102b)), through which fluid can exit into the recirculation conduit (218). The tank outlet (102b) may be positioned at a bottom end of the tank (102), such that fluid can automatically exit via gravitational force. The fluid can then return to the water source (206) and continuously re-circulate through the system. In some cases, a secondary pump is provided along the recirculation conduit (218), to promote fluid flow.

[0061] Various variations to the system (200) will now occur to the skilled artisan. For example, rather than the outlet (108a) - of the plasma reactor unit (108) - being coupled to the water conduit (216), the outlet (108a) can also feed directly into the water source (206). For example, the outlet (108a) may be inserted into the water source (206), and may directly treat the water source with an ACP jet. In this manner, the water tank (206) may function as a PAW generation tank, or a PAW source (206). This allows PAW (106) to exit the source (206), and travel directly - or through one or more conduits (216) - to the steeping tank (102).

[0062] Still further, the plasma reactor unit (108) may feed directly into the water inside the steeping tank (102). For example, as shown in FIG. 1, the outlet (108a) to the reactor unit (108) may be inserted through an opening (102') of the steeping tank (102).

[0063] To that end, the reactor unit (108) in systems (100) or (200) (FIGs. 1 and 2), may have any suitable design. For instance, as exemplified in FIG. 2, the reactor unit (108) may include a grounding electrode (202) and a high voltage (HV) electrode (204). The power supply (112) may include a ground wire (114a) coupled to the ground electrode (202), and a wire connected to the HV electrode (204). In other examples, the reactor unit (108) may be configured for other types of discharge, including corona or glow discharge.

[0064] In some examples, the system may include more than one plasma reactor unit (108). For example, in FIG. 1, multiple reactor units (108) can treat water, inside steeping tank (102), with ACP. In FIG. 2, multiple reactor units (108) can be coupled along the length of the water conduit (216) and/or disposed inside of the steeping tank (102) and/or water source (206).

IV. PAW CHARACTERISTICS.

[0065] In some embodiments, air or nitrogen may be used as the working gas to generate PAW having a significantly acidic pH. Nitrates and nitrites are produced in PAW, leading to the formation of nitrous and nitric acid. Also, acidic HiO ions could be formed by the reaction of hydrogen peroxide with water molecules, resulting in pH reduction. It has been previously reported that singlet oxygen plays a significant role in the acidification process. As singlet oxygen is involved in the production of nitrates, nitrites, and hydrogen peroxide, it is possible that the reduction in pH may not occur if the gases without oxygen and humidity are used for plasma generation. It has also been previously reported that the highest pH and lowest ORP were produced when nitrogen was used as the working gas. In some embodiments, after 30 min and 5 h storage of PAW, the pH was not changed which indicates the stability of the compounds causing the acidity of PAW during storage.

[0066] In other examples, other working gases may also be used to generate PAW, as known in the art. For example, these can include air, argon, or helium, or combinations thereof, as well as nitrogen and/or oxygen.

[0067] During the generation of PAW, using air as the working gas, it was found that the ORP of water increased from 194.4 to 400 mV after 30 min of ACP treatment of DW, demonstrating the greater oxidizing potential and reactivity of PAW compared to DW (Table 1). [0068] In an aqueous solution, ORP is the measure of the tendency of a solution to gain or lose electrons. A solution with a greater number of ORP has the tendency to gain electrons, i.e., to oxidize the other compounds. The reactive oxygen and nitrogen species (RONS) including hydrogen peroxide, ozone, nitrites, and nitrates are formed in the plasma phase above the surface of the water and diffused inside the water, contributing to the overall ORP of PAW.

RONS can also form inside the water. For example, once the plasma reactive species enters water, many new reactions can occur, which may include production of RONS.

[0069] The concentration of hydrogen peroxide was below the limit of the detection of the kits (LOD: 20 ppm) in DW and PAW (Table 1). Ozone is generated in the plasma phase above the surface of water by reaction R1 and then diffuses into the water. The concentration of ozone was 0.53 ppm in the PAW, and then the concentration decreased during storage due to its high reactivity. The dissolved ozone reacted with nitrite ions and hydrogen peroxide, resulting in the formation of nitrate ions and hydroxyl radical (R2-R3). Nitrates could be formed in the plasma and liquid phases. The PAW contained 11.95 ppm of nitrates which decreased in the first 30 min of storage and then remained the same during the 5 h storage period.

O+O ₂ ^O ₃ (Rl)

H ₂ O ₂ +O ₃ (aq.)^OH*+HO ₂ +O ₂ (R2)

NO ₂ +O ₃ (aq.) — NO ₃ +O ₂ (R3)

Table 1. Characteristics of plasma activated water H O Ozone Nitrate

PAW 30 min storage 3.88±0.04 ^b 392±28 ^a <20 0.12±0.08 ^b 8.66±1.4 ^b

PAW 5 h storage 3.88±0.03 ^b 372.7±8.1 ^a <20 0 ^c 8.65±1.4 ^b

Values are expressed as the mean ± standard deviation. Values with different letters in the same column are significantly different (p<0.05, n>3).

V. EXAMPLE METHOD.

[0070] FIG. 3 shows an example method (300) for PAW generation and treatment of cereal grains (e.g., NIB grains).

[0071] At (302), the plasma reactor unit (108) is operated to generate atmospheric cold plasma (ACP). For example, as shown in FIGs. 1 and 2, the reactor unit (108) may be operated and activated by coupling it to a power supply (112), or otherwise, tuming-on the power supply (112). The ACP is generated by feeding the reactor unit (108) with a gas source, e.g., pressurized air from the air tank (116).

[0072] At (304), the ACP is fed into water, to generate and produce plasma activated water (PAW). For example, as shown in FIG. 1, the plasma outlet (108a) can feed directly into the steeping tank (102), such that PAW is generated directly in the steeping tank where water is located. In other examples, as shown in FIG. 2, the plasma outlet (108a) can feed into a water conduit (216), feeding water between water source (206) and the steeping tank (102). In still other examples, the plasma outlet (108a) can feed directly into the water source (206) and/or steeping tank (102), in FIG. 2.

[0073] At (306), cereal grain (e.g., NIB) is steeped and exposed to the PAW. For example, as shown in FIGs. 1 and 2, the cereal grain is steeped inside of the steeping tank (102), which retains the PAW (106). [0074] In some examples, the water inside the steeping tank (102) may be treated with ACP, while the cereal grain is being steeped. That is, steps (304) and (306) may occur concurrently and/or partially concurrently. In other examples, the water is first treated with ACP to produce PAW, and thereafter, the ACP treatment is stopped and the cereal grain is steeped into the PAW.

[0075] In at least one example, the cereal grain is steeped in PAW after ACP treatment for at least 30 min, such as between about 30 minutes and 5 hours.

[0076] At (308), the cereal grain may be, in some cases, subject to air rest. For example, the cereal grain may be allowed to rest in air for an additional period of time, preferably at least about 1 hour, such as between about 1 hour and 24 hours.

VI, EXAMPLES.

[0077] The following examples are intended to exemplify specific embodiments of the invention.

(i.) Selected Barley Grains and Chemicals.

[0078] Naturally infected barley (NIB) grains (2-row malting variety named CDC Copeland) containing large concentrations of DON, with an initial moisture content of 10.27±0.04% (wet basis) were procured from Brandon Research and Development Centre, Agriculture and Agri -Food Canada, Brandon, Manitoba, Canada. The grains were kept in Ziploc bags and stored at 4°C until used. Analytical standard of DON (5 mg) was purchased from Milipore Sigma (Oakville, Ontario, Canada). HPLC-grade acetonitrile (ACN) was acquired from Fisher Scientific (Ottawa, Ontario, Canada).

(ii.) PAW Production and Characterization.

[0079] An ACP jet, generated by a plasma reactor unit (108) connected to a power supply (112) (PG 100-D, Advanced Plasma Solutions ™, Malvern, PA, USA), was used to treat 30 mL of distilled water (DW) beneath the water surface for 30 min to generate PAW (FIG. 1). [0080] Air was pumped, from an air tank (116), at a flow rate of 0.5 SLPM and the ACP jet was used at a frequency of 3500 Hz, 70% duty cycle, output voltage of 0-34 kV, current of 0-1 A, and a 10 ps pulse width in all the experiments.

[0081] The characteristics of PAW such as pH, ORP, ozone, nitrate, and H2O2 concentrations were determined. The pH, and ORP were measured using a pH meter (Fisher Scientific™, accumet AE150, Singapore) and ORP meter (Ohaus ™, ST20R, Parsippany, NJ, USA), respectively. CHEMets ™ kits (Midland, VA, USA) were used to measure ozone (K- 7404) based on DPD (N,N-diethyl-p-phenylenediamine) method; nitrate (K-6904) based on cadmium reduction method; and H2O2 (K-5510B) based on ferric thiocyanate method.

(Hi.) Determination of DON after PAW Treatments.

[0082] Ten grams of NIB grains were steeped in 30 mL PAW or DW (control) in a beaker for following treatments:

• Treatment A: steeping NIB in DW for 5 h followed by 19 h air rest;

• Treatment B: Steeping NIB in PAW for 5 h followed by 19 h air rest;

• Treatment C: 30 min ACP treatment of NIB inside DW followed by 4.5 h steeping, and 19 h air rest; and

• Treatment D: 30 min ACP treatment of NIB inside DW without further steeping or air rest.

[0083] At the end of steeping steps, the grains were drained for 5 min and then kept for 19 h air rest at 15±0.3°C, 76±4% RH. After air rest, the barley grain were dried at room temperature for 2 days. Then the DON content was measured using Reveal® Q+ test kits (Neogen, Lansing, MI, USA) based on the single-step lateral flow immunochromatographic assay. The limit of detection for the kits was 0.3 ppm and their specificity for DON was 100%. [0084] The kits were validated by quantifying DON by HPLC on a reversed-phase Agilent Zorbax ™ SB-C18 250 mm x 3 mm, 5-pm column with isocratic elution. HPLC was run at a wavelength of 218 nm using a photodiode array (PDA) detector with an injection volume of 25 pL and flow rate of 0.5 mL/min, using a mixture of water/ ACN/methanol (85: 12:3 v/v) as the mobile phase. My coSep™ 113 trichothecenes columns (Romer Labs, Tullin, Austria) were used for clean-up of mycotoxin from the matrix of grain following the manufacturer’s instructions.

(iv.) Water Uptake of Barley Treated by PAW.

[0085] Barley samples (1.00±0.02 g) were immersed in PAW or DW (control) at 22°C. Samples were taken at 1, 5, 24, 48 h to measure water uptake. Before weighing the samples, the grains were drained and the excessive water from the grain surface was removed using a filter paper. The increase in sample weight was considered as the water uptake of the grain.

(v.) Effect of PA W on Selected Quality Features of Green Malt.

[0086] Barley malt was produced by 2 days steeping using the treatments A, B, and C (as described in section 2.3) at 15±0.3°C and 76±4% environmental humidity followed by 3 days germination at 15°C, 92±4% RH. After each steeping cycle, the grains were drained for 5 min before air rest. Also, during 3 days germination process, the grains were washed by DW and drained followed by the addition of 3 mL DW to 10 g NIB each day.

[0087] The moisture content of the dry NIB grain and green malt was measured by the AACC Method using air oven drying for 2 h at 135°C (44-19.01, AACC International ™). The a-amylase, P-amylase, and P-glucanase activities in dry NIB grain and green malt were determined using K-MALTA 07/20 malt amylase assay kit and S-ABG100 03/11 malt and bacterial P-glucanase assay kit. Protein content of dry NIB grain and NIB grain before 3 day germination was determined by combustion using 6.25 as the nitrogen (%) to protein (%) conversion factor. [0088] Also, forty barley seeds in triplicate (40x3) were used for each treatment to determine the percentage of germinated acrospires and rootlets. The grains with growing acrospires and rootlets were considered as the germinated seed.

(vi.) Statistical Analysis.

[0089] SPSS (IBM SPSS v.27, Armonk, NY) was used for determining the significant differences (P<0.05) by one-way analysis of variance (ANOVA), followed by Duncan’s multiple range test. At least triplicate experiments were performed and the data were expressed as the mean ± standard deviation.

(vii.) Example Tests on the Effect of PA W on DON Reduction.

[0090] In tests, the initial DON content in NIB was 4.65 ppm, which is considered a heavily infected grain by fungal pathogen Fusarium graminearum. After the first wash and air rest during the steeping process using DW, the DON concentration was reduced by 33.3% (treatment A, Table 2). Using PAW, the reduction of DON was similar to that of DW after the first wash and air rest (treatment B). By immersing grain in DW and treating them with ACP to produce PAW for 30 min without any further steeping and air rest resulted in a 21.1% reduction of DON (treatment D).

[0091] During the steeping process, water absorption by grains causes an increase in moisture content. This process could lead to a change in the surface properties of the grains and hence affects the permeability of reactive species into the layers of the grain. It was likely the reason for a lower DON reduction after treatment D, where there was no steeping compared to treatments A and B (Table 2).

Table 2. Reduction of deoxy nival enol after steeping of naturally contaminated barley

Treatment A: Steeping NIB in DW for 5 h followed by 19 h air rest; Treatment B: Steeping NIB in PAW for 5 h followed by 19 h air rest; Treatment C: 30 min ACP treatment of NIB inside water followed by 4.5 h steeping, and 19 h air rest; Treatment D: 30 min ACP treatment of NIB inside water. Values are expressed as the mean ± standard deviation. Values with different letters in the same column are significantly different (p<0.05, n>4).

[0092] Treatment C resulted in the highest DON reduction in barley grains, with 58.4% reduction. Treatment C comprised treating NIB in water with ACP for 30 min followed by steeping and air rest. Without restriction to a theory, this may be result of interaction of shortlived reactive species such as hydroxyl radical (OH*), superoxide (02 ), and singlet oxygen ( ¹ 02) with the surface of the grains compared to treatments B and D.

[0093] It is believed that these changes in surface properties could lead to an increase in the diffusion of long-lived reactive species such as hydrogen peroxide, ozone, and nitrate into the inner layers of grains during the treatment of grain with PAW, thus reducing DON more than treatments B and D. It is likely that short-lived reactive species may directly interact with the seed coat and change the grain surface properties. Oxidation of the seed coat by RONS was reported previously, in which the short-lived reactive species could play a major role. Also, the hydroxyl radicals were reported to be responsible for cell wall loosening, enhancing the penetration of long-lived reactive species into the grain.

[0094] To that end, an increased reduction of DON may be achieved in treatment B by optimizing ACP treatment conditions. For example, this may involve using a larger plasma reactor (108) with higher voltages that produce a higher concentration of reactive species, such that steeping the grains - even after the ACP treatment is stopped - results in higher reductions to the DON. Likewise, for similar reasons, plasma reactors (108) with higher voltages may also result in greater reduction in DON in treatment D.

[0095] Another factor influencing the degradation of DON may be the pH of PAW. At acidic pH, especially at a pH range of 1-3, DON was previously reported in studies to be unstable and form an unknown degraded product. At pH 4, the degradation rate of DON was previously observed in studies at 11%. In disclosed embodiments, the average pH of PAW was observed at 3.87, which is believed to play a role in DON degradation. The DON content was not completely reduced since some small amount was likely residing in the inner layers of the grains, where the reactive species could not diffuse to the inner layers.

[0096] A significant (66%) reduction in DON content may be observed after simply pearling the barley grains, irrespective of the initial level of contamination. Accordingly, it is believed that DON is mainly present in the outer layer of the grain. After 120 seconds (s) pearling which reduced the grain mass by 47.7%, there was 7.9% DON left in the grain indicating how deep the DON could reside in the grains.

[0097] In a preferred embodiment, a malting process may benefit from using a PAW treatment, and particularly treatment C, as it results in greater DON degradation rate.

(viii.) Effect of PAW on Water Uptake, Germination, Enzymatic Activity, and Protein Content of Barley.

[0098] A conventional barley malting process includes three main steps: steeping, germination, and kilning. The water uptake and increase in water content initiates the germination of grains.

[0099] There appeared to be no significant difference between the water uptake and moisture content values of barley grains after first day of steeping, when using PAW or DW for all the steeping times (Table 3). [00100] Using PAW, the moisture content of the NIB increased after 2 ^nd day of steeping (Table 3). The moisture content of barley malt steeped in PAW (treatment B) was significantly (p<0.05) greater than that steeped in DW (treatment A) after second day of steeping. However, the moisture contents of steeped barley grains treated by PAW (treatment B) compared to direct ACP treatment (treatment C) after 1 ^st and 2 ^nd days of steeping were not significantly different (p>0.05).

Table 3. Effect of plasma-activated-water on the selected malting qualities

Treatment A Treatment B Treatment C Dry grain

Water uptake after 1 h (g water _d , _{8 9} , _{n 4} d absorbed / 100g) ¹⁸ - ^2±0 ' ^{6 18} - ^2±0 ' ⁴

Water uptake after 5 h (g water absorbed / 100g) ^o.i-i.y jo.y_z.z

Water uptake after 24 h (g water absorbed / 100g)

Water uptake after 48 h (g _{86 4±2 8} . water absorbed / 100g)

( ⁴ g ⁰ wate ^d r//w 10n0 ^t g ^ee s ^P a ^m m ⁸ plle) 34.2±1.6 ^a 35±0.2 ^a 33.7±0.7 ^a wate ² r//i 1 ^d n0n0 ^Y g ^S s ^t a ^C m ^CP p'l ⁿ ef) ^(S 41±1.8 ^b 45.3±2.5 ^a 43.5±1.7 ^ab

Germinated acrospire (%) 13.5±4.8 ^b 44.1±1.7 ^a 11.7±1.9 ^b

Germinated rootlets (%) 81.2±9.6 ^a 91.2±5.6 ^a 87.3±4.1 ^a a-amylase (units/g dry basis) 28.9±6.4 ^a 37.4±11.7 ^a 29.7±3.6 ^a 5.1±2.7 ^b

P-amylase (units/g dry basis) 23.9±3.7 ^b 27.6±1.2 ^a 28.4±1.6 ^a 22.6±4.1 ^b

P-glucanase (units/g dry basis) 238.3±45.2 ^a 257.5±79.8 ^a 246.4±41 ^a 129.7±34.7 ^b

Protein content (%) 14±0.7 ^a 13.6±0.2 ^a 14.2±0.5 ^a 13.5±0.2 ^a

Two days steeping using treatments A, B, C. Treatment A: Steeping NIB in DW for 5 h followed by 19 h air rest; Treatment B: Steeping NIB in PAW for 5 h followed by 19 h air rest; Treatment C: 30 min ACP treatment of NIB inside water followed by 4.5 h steeping, and 19 h air rest. For measuring the germination, enzymatic activity, and moisture content, treatments A, B, and C were followed by 3 day germination. Values are expressed as the mean ± standard deviation. Values with different letters in the same row are significantly different (p<0.05, n>4). [00101] Germination is an important step in malting, which is marked by embryo development, modification of endosperm, and the growth of rootlets and acrospire. The main visual change in the appearance of the grain during germination is the growth of rootlets and acrospire. At the end of the germination, maltsters prefer to have a germinated grain in which the acrospire length is approximately 75% of the kernel length. At the end of the germination period, the number of the germinated seeds based on their acrospire using treatment B was greater compared to treatment C and the control sample. However, there was no significant difference in the germination of the seeds based on their rootlets among all the treatments.

[00102] Also, for treating seeds with ACP, the working gas and the exposure time to ACP are the two important factors.

[00103] During germination different enzymes are produced in which three of the important ones are a-amylase, P-amylase, and P-glucanase. Lower levels of P-glucanase and higher levels of P-glucan (major constituent of barley endosperm cell wall) negatively affect the wort viscosity and beer filtration rate, thus reducing malting performance.

[00104] The a-amylase and P-glucanase activity values of NIB grain after treatments A, B and C were not statistically different, while P-amylase activity increased after treatments B and C compared to treatment A. The a-and P-amylases are the principal enzymes for hydrolyzing starch into fermentable sugars during malting. A higher enzymatic activity was observed in green malt compared to the non-malted grains in the previous studies. Enzymatic activity and the germination rate of the seeds treated by PAW can also be negatively affected due to oxidative stress if the characteristics of the PAW are not adjusted appropriately.

[00105] ACP can alter the enzymatic activity as it can affect the primary and secondary metabolites of plants and enhance their content. ROS and RNS were reported to promote seed dormancy release and subsequent germination in numerous plants. Also, nitric oxide, which is also generated in ACP, is an endogenous regulator of barley seed dormancy and increases germination.

[00106] Abscisic acid and gibberellic acid are the most important hormones regulating seed dormancy and germination. In barley, hydrogen peroxide, generated during ACP treatment, can activate gibberellic acid synthesis which results in the germination of the seed. All the aforementioned reactive species are generated via ACP and are present in PAW. Hence, it is more likely that they contribute to a greater germination rate of barley steeped in PAW. Depending on plasma treatment time and the working gas, our results concur with other studies which have reported increases in germination rates in ACP treated grains. These increases are likely due to the synthesis of more lytic enzymes.

[00107] The protein content of the NIB grains is not significantly affected by PAW treatment. The protein content is important during the malting process, as a strong protein- starch binding can limit the access of hydrolytic enzymes to starch and thus leading to a poor malt extract. However, a too low protein content can impair the brewing performance through poor yeast amino acid nutrition. Also, protein negatively increases haze formation in beer, but it is important for enhancing foam stability.

(ix.) Exemplary PA WB Reactor.

[00108] A custom designed continuous plasma activated water bubble (PAWB) reactor system was used to test its efficacy on DON reduction and germination improvement in naturally infested barley grains. This PAWB system, shown schematically in FIG. 2, used a plasma gas reactor and a venturi tube to continuously produce plasma bubbles in water and transported to a container, where the barley grains were steeped for specific periods. The barley grains were treated for 30 min in PAWB by continuous circulation of PAWB and followed by 4.5 hour steeping in the produced PAWB, and then 19 hour air rest.

[00109] The DON concentrations and germination of barley grains were determined after the steeping and air rest. There was a -10% increase in DON reduction and -5% improvement in germination of barley was observed when PAWB reactor system was used for steeping compared to distilled water. The DON reduction and germination rate could be further improved by longer treatment times and more number of recirculation of PAWB in the reactor.

Table 4. Use of a continuous plasma bubble reactor on DON reduction

Table 5. Characteristics of plasma activated water bubbles produced by continuous plasma reactor

Table 6. Use of a continuous plasma bubble reactor on germination of barley

VII. INTERPRETATION.

[00110] The corresponding structures, materials, acts, and equivalents of all means or steps 5 plus function elements in the claims appended to this specification are intended to include any structure, material, or act for performing the function in combination with other claimed elements as specifically claimed.

[00111] References in the specification to "one embodiment", "an embodiment", etc., indicate that the embodiment described may include a particular aspect, feature, structure, or 10 characteristic, but not every embodiment necessarily includes that aspect, feature, structure, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment referred to in other portions of the specification. Further, when a particular aspect, feature, structure, or characteristic is described in connection with an embodiment, it is within the knowledge of one skilled in the art to affect or connect such module, aspect, feature, 15 structure, or characteristic with other embodiments, whether or not explicitly described. In other words, any module, element or feature may be combined with any other element or feature in different embodiments, unless there is an obvious or inherent incompatibility, or it is specifically excluded.

[00112] It is further noted that the claims may be drafted to exclude any optional element. 0 As such, this statement is intended to serve as antecedent basis for the use of exclusive terminology, such as "solely," "only," and the like, in connection with the recitation of claim elements or use of a "negative" limitation. The terms "preferably," "preferred," "prefer," "optionally," "may," and similar terms are used to indicate that an item, condition or step being referred to is an optional (not required) feature of the invention.

[00113] The singular forms "a," "an," and "the" include the plural reference unless the context clearly dictates otherwise. The term "and/or" means any one of the items, any combination of the items, or all of the items with which this term is associated. The phrase "one or more" is readily understood by one of skill in the art, particularly when read in context of its usage.

[00114] The term "about" can refer to a variation of ± 5%, ± 10%, ± 20%, or ± 25% of the value specified. For example, "about 50" percent can in some embodiments carry a variation from 45 to 55 percent. For integer ranges, the term "about" or can include one or two integers greater than and/or less than a recited integer at each end of the range. Unless indicated otherwise herein, the term "about" is intended to include values and ranges proximate to the recited range that are equivalent in terms of the functionality of the composition, or the embodiment.

[00115] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges recited herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof, as well as the individual values making up the range, particularly integer values. A recited range includes each specific value, integer, decimal, or identity within the range. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, or tenths. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc.

[00116] As will also be understood by one skilled in the art, all language such as "up to", "at least", "greater than", "less than", "more than", "or more", and the like, include the number recited and such terms refer to ranges that can be subsequently broken down into sub-ranges as discussed above. In the same manner, all ratios recited herein also include all sub-ratios falling within the broader ratio. VIII. REFERENCES.

[00117] The following references are indicative of the level of skill of one skilled in the art, and are incorporated herein by reference in their entirety, where permitted. [1] Canadian Food Inspection Agency. (2017). Section 1 : Mycotoxins in Livestock Feed.

RG-8 Regulatory Guidance: Contaminants in Feed (formerly RG-1, Chapter 7). Retrieved from https://inspection.canada.ca/animal-health/livestock-feeds/r egulatory- guidance/rg-8/eng/1347383943203/1347384015909?chap=l