A TEMPERING COMPOSITION FOR TEMPERING GRAIN AND CONTROLLING PATHOGENS IN AND/OR ON SAID GRAIN, AN OXIDIZING COMPOSITION FOR PREPARING SAID TEMPERING COMPOSITION, A USE OF SAID TEMPERING COMPOSITION AND A METHOD OF USE OF SAID TEMPERING COMPOSITION

Abstract

A tempering composition for tempering grain in a tempering step and controlling pathogens susceptible to be present in and/or on said grain during the tempering step of the grain, said tempering step being eventually carried out before subjecting the tempered grain to a milling step; wherein said tempering composition comprises tempering-water and an oxidizing composition comprising at least one oxidizing agent and/or a precursor thereof, and eventually at least one an agriculturally acceptable excipient and/or at least one additive; and wherein the at least one oxidizing agent represents from 0.01 to 50% by weight of the oxidizing composition. A use and a method for tempering grain and controlling pathogens susceptible to be present on grain. An oxidizing composition for preparing the tempering composition. A use of the tempering composition for sanitizing mill systems. A method for sanitizing mill systems.

Claims

1-139. (canceled)

140. A tempering composition for tempering grain in a tempering step and controlling pathogens susceptible to be present in and/or on said grain during the tempering step of the grain, said tempering step being eventually carried out before subjecting the tempered grain to a milling step; wherein said tempering composition comprises tempering-water and an oxidizing composition comprising at least one oxidizing agent and/or a precursor thereof, and eventually at least one an agriculturally acceptable excipient and/or at least one additive; wherein the at least one oxidizing agent represents from 0.01 to 50% by weight of the oxidizing composition; herein the at least one oxidizing agent comprises: a) liquid peracid and/or in-situ generated peracid; and/or b) liquid hydrogen peroxide and/or hydrogen peroxide released from a hydrogen peroxide precursor; and wherein the tempering composition is to be applied to the grain at a rate varying from 20 to 80 liters of the tempering composition per ton of grain.

141. The tempering composition according to claim 140, wherein the oxidizing composition is admixed with the tempering-water in a weight ratio oxidizing composition:tempering-water varying from 4:16 to 2:78.

142. The tempering composition according to claim 140, wherein the tempering composition is suitable for contacting the grain for a period varying from 2 to 48 hours.

143. The tempering composition according to claim 140, wherein the at least one oxidizing agent comprises: a) a liquid peracetic acid and/or in-situ generated peracetic acid; b) optionally a liquid hydrogen peroxide and/or a hydrogen peroxide released from a hydrogen peroxide precursor; c) water; and c) optionally at least one additive and/or at least one agriculturally acceptable excipient.

144. The tempering composition according to claim 140, wherein the liquid peracetic acid is obtained from the reaction of acetic acid with hydrogen peroxide.

145. The tempering composition according to claim 1, wherein the food pathogens are selected from the group consisting of E. coli cocktail, Shiga toxin-producing E. coli (STEC), Verocytotoxin (VTEC), enterohemorrhagic E. coli (EHEC), Enterotoxigenic E. Coli (ETEC), Enteropathogenic E. coli (EPEC), Enteroaggregative E. coli (EAEC), Enteroinvasive E. coli (EIEC) and Diffusely adherent E. coli (DAEC).

146. The tempering composition according to claim 140, wherein the food pathogens are selected from the group consisting of Salmonella spp, Listeria spp., E. coli spp., Bacillus cereus, Campylobacter jejuni, Clostridium botulinum, Clostridium perfringens, Cryptosporidium, Cyclospora cayetanensis, Hepatitis A, Noroviruses, Shigella spp., Staphylococcus aureus, Vibrio parahaemolyticus and Vibrio vulnificus.

147. A method for tempering grain and for the control of pathogens susceptible to be present in and/or on said grain during a tempering step of the grain with a tempering composition to thereby obtain a tempered grain, said tempering step being eventually carried out before subjecting the tempered grain to a milling step; wherein said method comprises a step of contacting the tempering composition with the grain to thereby obtain the tempered grain, wherein the tempering composition comprises tempering-water and an oxidizing composition comprising at least one oxidizing agent and/or a precursor thereof, and eventually at least one an agriculturally acceptable excipient and/or at least one additive; wherein the at least one oxidizing agent represents 0.01 to 50% by weight of the oxidizing composition; wherein the at least one oxidizing agent comprises: a) liquid peracid and/or in-situ generated peracid; and/or b) liquid hydrogen peroxide and/or hydrogen peroxide released from a hydrogen peroxide precursor; and wherein the tempering composition is applied to the grain at a rate varying from 20 to 80 liters of the tempering composition per ton of grain.

148. The method according to claim 147, wherein the oxidizing composition is admixed with the tempering-water in a weight ratio oxidizing composition:tempering-water varying from 4:16 to 2:78.

149. The method according to claim 147, wherein the tempering-water, the oxidizing composition comprising the at least one oxidizing agent and/or a precursor thereof, and eventually the at least one an agriculturally acceptable excipient and/or further at least one additive, are mixed together in a mixing apparatus.

150. The method according to claim 147, wherein the tempering composition is contacted with grain in the tempering step by pumping, fumigating, spraying, misting or vaporizing said tempering composition on the grain.

151. The method according to claim 150, wherein a flow of the tempering composition is sprayed on a flow of the grain, and then the mixture of the grain and the tempering composition is held in a tempering tank for a period varying from 2 to 48 hours.

152. The method according to claim 147, wherein the at least one oxidizing agent comprises: a) a liquid peracetic acid and/or an in-situ generated peracetic acid; b) optionally a liquid hydrogen peroxide and/or a hydrogen peroxide released from a hydrogen peroxide precursor; c) water; and d) optionally at least one additive and/or at least one agriculturally acceptable excipient.

153. The method according to claim 147, wherein the liquid peracetic acid obtained from the reaction of acetic acid with hydrogen peroxide.

154. The method according to claim 147, wherein the food pathogens are selected from the group consisting of E. coli cocktail, Shiga toxin-producing E. coli (STEC), Verocytotoxin (VTEC), enterohemorrhagic E. coli (EHEC), Enterotoxigenic E. Coli (ETEC), Enteropathogenic E. coli (EPEC), Enteroaggregative E. coli (EAEC), Enteroinvasive E. coli (EIEC) and Diffusely adherent E. coli (DAEC).

155. The method according to claim 147, wherein the food pathogens are selected from the group consisting of Salmonella spp, Listeria spp., E. coli spp., Bacillus cereus, Campylobacter jejuni, Clostridium botulinum, Clostridium perfringens, Cryptosporidium, Cyclospora cayetanensis, Hepatitis A, Noroviruses, Shigella spp., Staphylococcus aureus, Vibrio parahaemolyticus and Vibrio vulnificus.

156. The method according to claim 147, wherein during the subsequent milling step the tempering composition further cleans and sanitizes mill systems to prevent cross-contamination and reduce the load of microorganisms in said mill systems.

157. An oxidizing composition useful for the preparation of a tempering composition as defined in claim 140, wherein the oxidizing composition comprises at least one oxidizing agent and/or a precursor thereof, and eventually at least one an agriculturally acceptable excipient and/or at least one additive; and wherein the at least one oxidizing agent represents from 0.01 to 50% by weight of the oxidizing composition; wherein the at least one oxidizing agent comprises: a) liquid peracid and/or in-situ generated peracid; and/or b) liquid hydrogen peroxide and/or hydrogen peroxide released from a hydrogen peroxide precursor; and wherein the tempering composition is to be applied to the grain at a rate varying from 20 to 80 liters of the tempering composition per ton of grain.

158. A method for sanitizing mill systems, said method comprising a step of contacting the mill systems with the tempering composition defined in claim 140.

159. A method for sanitizing mill systems, said method comprising a step of contacting the mill systems with the oxidizing composition defined in claim 157.

Description

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

[0311] There will be provided hereinafter experimental tests illustrating particularly preferred embodiments of the invention

Protocols, results and conclusions

Example 1

Introduction

[0312] An organic flour mill was selected in Quebec Canada to conduct a trial in order to determine the efficacy of the organic novel composition identified as Neo-Temper which is composed of a stabilized peracetic acid generated by the reaction of acetic acid and hydrogen peroxide. More particularly this Neo-Temper tailored for the tempering-holding-milling phases in flour mills was compared to citric and lactic acids as antibacterial agents. Neo-Temper has the following formulation:

TABLE-US-00001 Percentage Ingredients CAS # (w/w) Distilled Water 7732-18-5 34.5% Acetic Acid 92% Post-reaction: 64-19-7 13.5% 5% Hydrogen Generates 7722-84-1 50.0% peracetic Peroxide 50% guaranteed acid minimum 50,000 ppm active pre-formed peracetic acid (CAS #79-21-0) Sulphuric Acid 96% 7664-93-9 1.1% Dequest 2010: 2809-21-4 0.9% 1-hydroxyethylidene- 1,1-diphosphonic acid TOTAL: 100% w/w
Guarantee: Minimum 5% active

Protocol

[0313] Tempering of wheat is obtained by adding water to the wheat at a rate to reach the desired moisture content of wheat. Concerning the following experiments, a determined volume of Neo-Temper was substituted to a corresponding volume of the tempering-water (e.g. if 20 liters of tempering are normally required for the tempering of wheat, then the tempering is achieved with a mix of 18 liters of water and 2 liters an oxidizing composition (i.e. 2 liters of Neo-Temper). The holding time for the following experiments was 16 hours before milling, and the usual amount of tempering-water (before any partial substitution with an oxidizing composition) is about 40 liters/tonne of wheat. [0314] Treatment 1—Neo-Temper 2 L: 2 Liters of Neo-Temper (substituting 2 L water) were added per ton of wheat during water-addition in the tempering step. [0315] Treatment 2—Neo-Temper 4 L: 4 Liters of Neo-Temper (substituting 4 L water) were added per ton of wheat during water-addition in the tempering step. [0316] Treatment 3—Neo-Temper+Surfactant 4 L: 4 Liters of Neo-Temper containing an organic surfactant (substituting 4 L water) were added per ton of wheat during water-addition in the tempering step. The surfactant is Tween 20 (a polysorbate surfactant). [0317] Treatment 4—Citric acid: 2 Kg of Citric acid were added per ton of wheat during water-addition in the tampering step. [0318] Treatment 5—Lactic acid: 2 Liters of lactic acid (substituting 2 L water) were added per ton of wheat during water-addition in the tempering step. [0319] Treatment 6—water: Only water was added as usual with no organic antimicrobial solution.

[0320] The results are described in Table 1.

TABLE-US-00002 TABLE 1 TRT 1 TRT 2 TRT 3 Neo- Neo- Neo-Temper TRT 4 TRT 5 TRT6 Temper Temper 4L + Citric Lactic Control 2L 4L surf. acid acid H.sub.2O FOSS % Proteins 11.38 11.46 11.34 11.32 11.54 11.37 % humidity 13.1 13.2 13.4 13.5 12.8 12.9 % Ash 0.49 0.52 0.49 0.48 0.51 0.48 % gluten 31.62 31.17 31.56 31.97 31.83 31.91 % absorption 59.98 60.49 59.8 59.82 60.85 60.68 IDC second 346 348 366 367 338 356 MIXOLAB % H.sub.2O 59.9 58.8 60.1 60.1 59.6 60.2 Stability (min) 14 21.5 15 13.5 17.5 13.5 Aff 12 min (Nm) 0.005 0.016 0.018 0.022 0.022 0.01 PMT (second) 214 178 202 185 186 176 GPT BEM (Brabender 54 51 51 51 52 54 unit) AM/BM (second) 48 47 47 46 45 49 PM/AM (second) 48 42 46 45 42 48 % Gluten—Wet 35.4 34.6 34.6 34.8 34.6 35.7 % Protein content 11.6 11.2 11.2 11.3 11.2 11.7 % Hydration 60 59 60 60 59.5 60 VMI Time (second) 54 62 54 54 43 55 Temperature (° C.) 24.8 24 22.5 23.6 22.6 23.8 Strength (Watt per 517 510 493 510 509 520 hour) Energy (Watt per 15.6 16.8 7.8 15.8 14.9 8.8 hour) EXTENSO 1 Energy (cm-2) 134 126 131 130 148 139 Resistance (EU) (EU = extensograph 458 440 429 459 421 472 petrin outle tunit) Extensibility (mm) 154 155 158 149 169 156 Maximum (mm) 700 652 658 705 712 720 Ratio (EU/mm) 3 2.8 2.7 3.1 2.5 3 Ratio Max (EU/mm) 4.5 4.2 4.2 4.7 4.2 4.6 EXTENSO 2 Energy (cm-2) 141 125 141 136 124 128 Division Resistance (EU) 744 686 667 613 532 636 after fermen Extensibility (mm) 125 120 135 136 136 127 Maximum (mm) 926 880 858 809 734 861 Ratio (EU/mm) 6 5.7 4.9 4.5 3.9 5 Ratio Max (EU/mm) 7.4 7.3 6.4 6 5.4 6.8 EXTENSO 1 Energy (cm-2) 80 82 103 75 64 74 3 h 30 after Resistance (EU) 240 257 222 207 200 240 fermen. Extensibility (mm) 180 185 217 193 179 174 Maximum (mm) 329 319 360 298 270 303 Ratio (EU/mm) 1.3 1.4 1 1.1 1.1 1.4 Ratio Max (EU/mm) 1.8 1.7 1.7 1.5 1.5 1.7 size poolish cm 13.8 13.8 14.1 14.2 15.1 12.2 Bread size- mm 157 165 132 158 146 149 mold

[0321] Conclusion: Neo-Temper (2 L per Ton) had no impact on flour functionality. Neo-Temper (4 L per Ton) was accepted by the organic mill but borderline. Neo-Temper+surfactant (i.e. Tween 20, a polysorbate surfactant) was rejected for its effect on energy, resistance and extensibility of the bread. Likewise, citric acid and Lactic acid were both rejected for their negative impact on resistance. Moreover, Neo-Temper 2 L per ton and 4 L per ton had no negative impact on sensory and physical characteristics.

[0322] For those skilled in the art, it was unusual that both citric and lactic acid (2 kg and 2 L per ton respectively) had a negative impact on functionality whereas Neo-Temper 2 L and even up to 4 L per ton of wheat) had no negative impact on functionality noting that Neo-Temper contains not only an acid component like citric and lactic acid but it contains a strong acidic oxidizer (hydrogen peroxide) at high percentage.

[0323] Also, for those skilled in the art, it was unusual that Neo-Temper 2 L and even up to 4 L per ton of wheat) had no negative impact on sensory noting that Neo-Temper has a strong acetic acid (Vinegar) smell.

[0324] Moreover, there is no drying step before milling which usually helps in odor removal. The presence of peracetic acid and hydrogen peroxide did not bleach or discolor the wheat and had no negative effect on the physical characteristics and taste.

[0325] Finally, for those skilled in the art, it was unusual that the surfactant (i.e. Tween 20, a polysorbate surfactant) had a such negative impact on gluten and extensibility.

[0326] Also, it was surprising for those skilled in the art that oxidizing agents such as peracetic acid and hydrogen peroxide did not ruin the food (sensory, functionality) and that the hold time during the tempering phase helps in the reduction of microorganisms but did not damage the food. It was also surprising that hydrogen peroxide performed better than other oxidizing agents and acids.

[0327] Also, it was surprising for those skilled in the art that this composition provides control of pathogens while maintaining functionality. That was not obvious to a person of ordinary skill in the art. It was surprising that the flour and bran do not smell like vinegar. The fact that it is rate-sensitive and that 4 L of the composition hurts functionality, while 2-3 L does not is also surprising.

Example 2

Introduction

[0328] Since Neo-Temper 2 L per ton had no negative impact on functionality, physical characteristics and sensory whereas Neo-Temper 4 L per ton was borderline, the objective of this experiment was to repeat the treatments and include Neo-Temper 3 L per ton.

Protocol

[0329] Concerning the following experiments, a determined volume of Neo-Temper was substituted to a corresponding volume of the tempering-water (e.g. if 20 liters of tempering are normally required for the tempering of wheat, then the tempering is achieved with a mix of 17 liters of water and 3 liters an oxidizing composition (i.e. 3 liters of Neo-Temper). The for the following experiments was 16 hours before milling, and the usual amount of tempering-water (before any partial substitution with an oxidizing composition) was about 20 liters/tonne of wheat. More particularly, a flow of the Neo-Temper was directly injected with a metering pump (ProMinent® into the flow of tempering-water going to tempering bins. A total solution of 20 L including the tempering-water and the oxidizing composition (New-Temper) were sprayed on 1 ton of wheat in the tempering bins. [0330] Treatment 1—Neo-Temper 2 L: 2 Liters of Neo-Temper (substituting 2 L water) were added per ton of wheat during water-addition in the tampering step. [0331] Treatment 2—Neo-Temper 3 L: 3 Liters of Neo-Temper (substituting 3 L water) were added per ton of wheat during water-addition in the tempering step. [0332] Treatment 3—water: Only water was added as usual with no organic antimicrobial solution.

[0333] The results are described in Table 2.

TABLE-US-00003 TABLE 2 TRT 1- TRT 2- TRT 3- 20.06.2017 20.06.2017 20.06.2017 Control 2LNeo- 3L Neo- Temper Temper 11.25 11.28 11.11 % proteins FOSS 14.92 14.26 14.6 % humidity 0.47 0.52 0.43 % Ash 29.79 30.7 31.04 % gluten 59.41 59.33 58.95 % absorption 342 362 355 second IDC 58.6 % H.sub.2O MIXOLAB 19.5 Stability (min) 0.065 Weakening 12 min (Nm). 58.6 58.6 58.6 % H.sub.2O MIXOLAB+ −0.086 −0.1 −0.102 Alpha (Nm/min) 0.432 0.374 0.368 Beta (Nm/min) 0 −0.012 −0.03 Y (Nm/min) 169 174 162 PMT (second) GPT 51 52 52 BEM (Brabender unit) 35 47 37 AM/BM (second) 46 44 45 PM/AM (second) 34.9 35 34.7 % Gluten Humid 11.3 11.4 11.3 % proteins

[0334] Conclusion: Neo-Tem per (2 L and 3 L per Ton) had no impact on functionality. The effect of Neo-Temper (2 L and 3 L per ton) is almost similar to the untreated as shown in Table 2. Moreover, Neo-Temper (2 L and 3 L per ton) had no negative impact on sensory and physical characteristics.

Example 3

Introduction

[0335] Since Neo-Temper (2 L and 3 L per ton) had no impact on functionality, sensory and physical characteristics, it was worth investigating the efficacy of Neo-Temper in reducing E. coli strains on wheat.

Protocol:

Preparation of Inoculant:

[0336] 3 strains of E. coli O157:H7 were each grown to stationary phase in 50 mL BHI broth (35C with shaking at 150 rpm, 18-24 h). Cells were pelleted by centrifugation at 4,000×g for 10 minutes and washed 3× with 50 mL of 0.1% peptone water. Cells were resuspended in 8 mL of 0.1% peptone water and pooled to obtain 2 inoculum cocktails for O157:H7 strains and for O121 strains. The estimated cell concentration was 3.6E+08 cfu/mL for O157:H7.

[0337] Sample Inoculation

[0338] 2 mL of the inoculum was mixed with 100 g of Wheat with vigorous mixing and allowed to dry/acclimatize (exposed to air) for 24 hours in a biohood cabinet at 22C, Followed by treatment or no treatment of the spiked samples with the various solutions. Samples were analysed 24 hours after treatment.

Enumeration

[0339] Samples were mixed with 1:2 ratio of sample to mTSB enrichment broth. Additional 10-fold serial dilutions from the 1/2 dilution were prepared in 0.1% peptone water. For enumeration, 1 mL (0.33 ml×3 plates) from the 1/2 dilution and 0.1 mL of subsequent 10-fold dilutions were spread plated onto Rainbow Agar. Enumeration results are reported in cfu/g (the minimum detection limit was 2 cfu/g). For presence/absence the initial 1/2 diluted sample in mTSB was enriched at 42° C. for 18-24 hours, 1 mL was then subjected to immunomagnetic separation using O157 coated magnetic beads and plated onto ChromAgarO157 and CR-SMAC for O157:H7 inoculated samples. Suspect colonies were confirmed via the use of antisera or PCR.

[0340] Samples used in this trial were initially negative for the presence of STEC as determined by PCR and thus were considered to be free of the target organism prior to inoculation. Results are reported in Table 3.

TABLE-US-00004 TABLE 3 Efficacy of Neo-Temper on wheat infected with STEC Treatments Grain CFU/ml WHEAT O157 UT-1 Wheat 680000 WHEAT O157 UT-2 Wheat 360000 WHEAT O157 UT-3 Wheat 152000 WHEAT O157 4 L/ton A-1 Wheat <2 WHEAT O157 4 L/ton A-2 Wheat <2 WHEAT O157 4 L/ton A-3 Wheat <2 WHEAT O157 4 L + surf/ton Wheat <2 B-1 WHEAT O157 4 L + surf/ton Wheat <2 B-2 WHEAT O157 4 L + surf/ton Wheat <2 B-3 WHEAT O157 2 L/ton C-1 Wheat 2 WHEAT O157 2 L/ton C-2 Wheat 4 WHEAT O157 2 L/ton C-3 Wheat <2

Conclusion:

[0341] Growth potential of E. coli of serotype O157 on artificially inoculated Wheat and treated with three Neo-Temper formulations at a rate: 4 mL/100 g (A), 4 mL+surf/100 g (B) and 2 mL/100 g (C) sample with a 24 hour hold period compared to untreated controls. This experiment was done in triplicate. The Untreated O157:H7 control was 5.52 log cfu/g. Below are the results of treatments A, B and C.

A) Growth potential of E. coli O157:H7 on Wheat treated with Neo-Temper (4 L) mixed in 36 L water per ton wheat was −5.22 to −5.52 log cfu/g
B) Growth potential of E. coli O157:H7 on Wheat treated with Neo-Temper (4 L) containing 0.2 L surfactant (i.e. Tween 20, a polysorbate surfactant) mixed in 36 L water was −5.22 to −5.52 log cfu/g
C) Growth potential of E. coli O157:H7 on Wheat treated with Neo-Temper (2 L) mixed in 38 L water was −5.07 log cfu/g

Example 4

[0342] In the following example, the following oxidizing compositions were used: Neo-Temper and Neo-Temper with hydrogen peroxide. Those oxidizing composition have the following formulations:

Neo-Temper has the following formulation:

TABLE-US-00005 Ingredients CAS # Percentage (w/w) Distilled Water 7732-18-5 34.5% Acetic Acid 92% Post-reaction: 64-19-7 13.5% 5% Hydrogen Generates 7722-84-1 50.0% peracetic Peroxide guaranteed acid minimum 50,000 ppm active pre-formed peracetic acid (CAS #79-21-0) Sulphuric Acid 96% 7664-93-9 1.1% Dequest 2010: 2809-21-4 0.9% 1-hydroxyethylidene- 1,1-diphosphonic acid TOTAL: 100% w/w
Guarantee: Minimum 5% active
Neo-Temper+hydrogen peroxide

[0343] The above-mentioned Neo-Temper formulation and 35% hydrogen peroxide were simultaneously injected with a metering pump (ProMinent®) into a flow of tempering-water going to tempering bins.

[0344] Efficacy of Neo-Temper Against Shiga-Toxin Producing E. coli (STEC) and its Non-Pathogenic Surrogate on Wheat

[0345] The efficacy of “Neo-Temper” and “Neo-Temper with hydrogen peroxide” was tested in the following experiments: [0346] 1. Pathogen challenge test—Efficacy of Neo-Temper (with 16 h hold time post-treatment) in eliminating a cocktail of STEC Escherichia coli on wheat [0347] 2. Surrogate compatibility test—Efficacy of Neo-Temper (with 8 h hold time post treatment) in eliminating a Shiga-toxin producing Escherichia coli cocktail compared to a non-pathogenic E. coli cocktail on irradiated wheat [0348] 3. Test on STEC surrogate—Efficacy of Neo-Temper with hydrogen peroxide (with 16 h hold post-treatment) in eliminating a non-pathogenic E. coli cocktail (STEC surrogate) on wheat

Procedure:

[0349] 1. Pathogen challenge test:

[0350] Wheat samples (200 g each) were inoculated with a seven strain cocktail of shiga-toxin producing E. coli (STEC) (serotypes O26:H11, O103:H2, O11:NM, O121:H19, O145:NM, 045:H2 and O157:H7) each grown in BHI broth (35° C. with shaking at 150 rpm, for 18-24 h). Cultures were pooled and cells were pelleted by centrifugation at 4,000 g for 10 minutes and washed 3 times with 0.1% peptone water. Cells were resuspended in 0.1% peptone water. Two mL of the cocktail inoculum was vigorously mixed with 100 g of wheat (N=5) and allowed to dry/acclimatize (exposed to air) for 24 hours in a biohood cabinet at 22° C., followed by treatment or no treatment of inoculated samples with various solutions. Treated samples were held in biosafety cabinet at 22° C. for 16 h.

[0351] The solutions tested were: [0352] Solution A: 2 L Neo-Temper in 21 L H.sub.2O (applied at 23 L/tonne): [0353] Solution B: 2 L Neo-Temper in 68 L H.sub.2O (applied at 70 L/tonne) [0354] Solution C: 3 L Neo-Temper in 20 L H.sub.2O (applied at 23 L/tonne) [0355] Solution D: 3 L Neo-Temper in 67 L H.sub.2O (applied at 70 L/tonne)

[0356] More particularly, the Neo-Temper was injected with a metering pump (ProMinent®) into tempering-water, and then sprayed via nozzles on wheat and held in tempering bins for at least 4 hours.

[0357] The E. coli counts in CFU/g of treated or untreated samples were obtained as follows: samples were first homogenized/mixed with 1:2 ratio of sample to 0.1% peptone water and stomached for 2 min. Additional 10-fold serial dilutions from the 1/2 dilution were also prepared in 0.1% peptone water. For enumeration, 1 mL (0.33 ml×3 plates) from the 1/2 dilution and subsequent 10-fold dilutions were spread plated onto TSA. Enumeration results are reported in CFU/g (the minimum detection limit was 2 CFU/g). This experiment was repeated independently three times for a total of three biological replicates with 5 replicates in each biological replicate (total N=15). Enumeration results are reported in log CFU/g (minimum detection limit of 2 CFU/g). Samples used were initially negative for the presence of STEC as determined by PCR and thus were considered free of the target organism prior to inoculation.

2. Surrogate compatibility test:

[0358] Irradiated wheat samples (200 g each) were inoculated with either 1) a seven strain cocktail of shiga-toxin producing E. coli (STEC) (serotypes O26:H11, O103:H2, O11:NM, O121:H19, O145:NM, 045:H2 and O157:H7) or 2) a non-pathogenic E. coli cocktail composed of ATTC strains (BAA-1427, BAA-1428, BAA-1429, BAA-1430 and BBA-1431) each grown in BHI broth (35° C. with shaking at 175 rpm, for 18-24 h). Cultures were pooled and cells were pelleted by centrifugation at 4,000 g for 10 minutes and washed 3 times with 0.1% peptone water. Cells were resuspended in 0.1% peptone water. Two mL of the cocktail inoculum was vigorously mixed with 100 g of wheat (N=3) and allowed to dry/acclimatize (exposed to air) for 24 hours in a biohood cabinet at 22° C., followed by treatment or no treatment of inoculated samples with 7 mL/100 g (70 L/tonne) of the solution D (3 L of Neo-Temper:67 L of water). Treated samples were held in biosafety cabinet at 22° C. for 8 h. E. coli counts in CFU/g of treated or untreated samples were obtained as follows: Samples were first homogenized/mixed with 1:2 ratio of sample to 0.1% peptone water and stomached for 2 min. Additional 10-fold serial dilutions from the 1/2 dilution were also prepared in 0.1% peptone water. For enumeration, 1 mL (0.33 ml×3 plates) from the 1/2 dilution and subsequent 10-fold dilutions were spread plated onto TSA. Enumeration results are reported in CFU/g (the minimum detection limit was 2 CFU/g). This experiment was repeated independently three times (N=3). Enumeration results are reported in log CFU/g (minimum detection limit of 2 CFU/g). The wheat utilized in these trial was confirmed to be free of background microbiota.

3. Test on STEC surrogate:

[0359] Wheat samples of 2 kg were inoculated with a non-pathogenic E. coli cocktail composed of ATTC strains (BAA-1427, BAA-1428, BAA-1429, BAA-1430 and BBA-1431). The strains were grown separately for 18 hours at 35° C. and mixed the day of the inoculation. The inoculum was applied while mixing the grain vigorously in a kitchen mixer at a rate of 30 mL per kg of wheat, and allowed to dry/acclimatize (exposed to air) for 24 hours in a biohood cabinet at 22° C. Then, 3 samples of 45 g each, were withdrawn for enumeration. These constituted an analysis of untreated controls (UTC) to determine the concentration of surviving bacteria before treatment. The remaining wheat was treated at a rate of 70 L/tonne of a tempering composition (3 L of Neo-Temper:10 L of 35% hydrogen peroxide:57 L of water). After 16 hour of the treatment, 1 kg of the treated wheat was milled into flour in a laboratory scale miller, and 3 samples of flour, 45 g each, were taken for E. coli enumeration. In addition, the flour samples were stored and E. coli enumeration was performed at 1, 2 and 4 weeks after the treatment. This experiment was repeated independently three times for a total of three biological replicates with 5 replicates in each biological replicate (total N=15). E. coli counts in CFU/g of treated or untreated samples were obtained as follows: Samples were first homogenized/mixed with 1:2 ratio of sample to 0.1% peptone water and stomached for 2 min. Additional 10-fold serial dilutions from the 1/2 dilution were also prepared in 0.1% peptone water. For enumeration, 1 mL (0.33 ml×3 plates) from the 1/2 dilution and subsequent 10-fold dilutions were spread plated onto Brilliance E.coli coliform selective agar. Enumeration results are reported in CFU/g (the minimum detection limit was 2 CFU/g).

TABLE-US-00006 Effect of Neo-Temper (with 16 hour Hold Post-treatment) on STEC Cocktail Inoculated on Wheat Effect of Neo-Temper (with 16 hour Hold Post-treatment) on STEC Cocktail Inoculated on Wheat Recovered STEC population Average Log (Avg.,N = 5 Each trial) (log CFU/g ± SD) (log CFU/g ± Reduction Sample Trial #1 Trial #2 Trial #3 SD) (log CFU/g) UTC 6.54 ± 5.53 ± 5.63 ± 5.90 ± N/A 0.29 0.11 0.23 0.55 Solution 3.17 ± 2.57 ± 3.07 ± 2.94 ± 2.96 A 23 L/tonne (2 L Neo- Temper 0.28 0.43 0.06 0.32 in 21 L H.sub.2O) Solution 3.75 ± 2.54 ± 2.51 ± 2.93 ± 2.97 B 70 L/tonne 0.13 0.14 0.18 0.71 (2 L Neo- Temper in 68 L H.sub.2O) Solution 3.20 ± 3.50 ± 3.18 ± 3.29 ± 2.61 C 23 L/tonne 0.13 0.08 0.16 0.18 (3 L Neo- Temper in 20 L H.sub.2O) Solution 3.04 ± 2.06 ± 2.46 ± 2.52 ± 3.38 D 70 L/tonne 0.17 0.28 0.27 0.49 (3 L Neo- Temper in 67 L H.sub.2O) SD = standard deviation; UTC = untreated control (inoculated)

Results

1. Pathogen Challenge Test:

[0360] The table below shows the efficacy of different solutions against a seven-strain STEC cocktail. The reported initial level of STEC (5.90±0.55 Log CFU/g) belongs to the untreated wheat samples that were enumerated 24 hour after inoculation. The results show that population of STEC dropped to 2.94±0.32, 2.93±0.71, 3.29±0.18 and 2.52±0.49 log CFU/g after treatment with solutions A, B, C, or D, respectively, producing 2.96, 2.97, 2.61, and 3.38 log reduction on STEC. Solution D (3 L Neo-Temper in 67 L H.sub.2O), applied at 70 mL/kg (70 L/tonne), was the most effective against STEC. Therefore it was chosen for future testing.

2. Surrogate Compatibility Test:

[0361] The table below shows the comparison of the efficacy of a tempering composition (3 L of Neo-Temper:67 L of water), on a seven-strain STEC cocktail and a five strain cocktail of non-pathogenic E. coli on wheat. The initial counts of the non-pathogenic E. coli cocktail were about 0.7 log CFU/g lower than the STEC cocktail. However, the average log CFU/g reduction values (calculated as the difference between the average UTC log CFU/g and the treated sample's log CFU/g), were comparable after 8 h treatment with Neo-Temper, with no statistically significant difference (P>0.05) determined by one-way ANOVA (alpha=0.05).

TABLE-US-00007 Effect of tempering composition ((3 L of Neo-Temper: 67 L of water) at 70 L/tonne with 8 hour Hold Post-treatment) on an STEC Cocktail and a Non-Pathogenic E. coli cocktail on Wheat Effect of tempering composition ((3 L of Neo-Temper: 67 L of water) at 70 L/tonne with 8 hour Hold Post-treatment) on an STEC Cocktail and a Non-Pathogenic E. coli cocktail on Wheat Average Average Log Recovered ± SD Reduction Sample (log CFU/g) (log CFU/g) Wheat inoculated with 6.29 ± 0.19 — STEC cocktail, UTC Wheat inoculated with 4.20 ± 0.31 2.09 STEC cocktail, Treated Wheat inoculated with non- 5.60 ± 0.31 — pathogenic E. coli cocktail, UTC Wheat inoculated with non- 3.64 ± 0.31 1.96 pathogenic E. coli cocktail, Treated SD = standard deviation; UTC = untreated control (inoculated)
3. Test on STEC surrogate:

[0362] The table below shows the efficacy of a tempering composition (3 L of Neo-Temper:10 L of 35% hydrogen peroxide:57 L of water) against a the non-pathogenic E. coli cocktail, which previously demonstrated to be a suitable surrogate for STEC on wheat. The treatment was able to achieve 3.36 log CFU/g reduction on the E. coli cocktail on the flour obtained from the treated wheat, after 16 h hold. In addition, the counts of E. coli in the flour decreased consistently after 1, 2 and 4 weeks of the treatment, achieving 4.11, 4.70 and 5.31 log CFU/g reductions, respectively

TABLE-US-00008 Effect of the tempering composition ((3 L of Neo-Temper:10 L of 35% hydrogen peroxide:57 L of water) at 70 L/tonne, with 16 hour Hold Post-treatment) on a Non-Pathogenic E. coli cocktail on Wheat Effect of the tempering composition ((3 L of Neo-Temper: 10 L of 35% hydrogen peroxide: 57 L of water) at 70 L/tonne, with 16 hour Hold Post-treatment) on a Non-Pathogenic E. coli cocktail on Wheat WHEAT Average Average Untreated Log Control FLOUR Reduction SD (log Average Recovered (log Sample CFU/g) SD (log CFU/g) CFU/g) Week 0 (16 h hold) 5.82 ± 0.21 2.46 ± 0.11 3.36 Week 1 NA 1.70 ± 0.37 4.11 Week 2 0.55 ± 0.24 4.70 Week 4 0.84 ± 0.53 5.31 SD = standard deviation

CONCLUSIONS

[0363] The pathogen challenge test demonstrated that Solution D (3 L of Neo-Temper:67 L of water) applied at 70 L/tonne, was the most effective against the STEC cocktail, achieving 3.38 log CFU/g reduction with 16 h hold time post-treatment. Therefore, this formula and rate were used for the rest of the tests.

[0364] The surrogate compatibility test proved that the non-pathogenic cocktail of E. coli is a suitable surrogate for STEC for testing the efficacy the tempering composition (3 L of Neo-Temper:67 L of water) on wheat, as indicated by the consistency in the log CFU/g reductions achieved 8 h after the treatment at 70 L/tonne.

[0365] The efficacy of the tempering composition (3 L of Neo-Temper:10 L of 35% hydrogen peroxide:57 L of water) applied at 70 L/tonne on the STEC surrogate cocktail on wheat was demonstrated by achieving >3 log CFU/g reduction 16 h after the treatment and by the increase in the log CFU/g reduction to >5, after 4 weeks of the treatment.

[0366] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the present invention and scope of the appended claims.