A method for providing a mixture of grain and a determined population of at least one pathogenic or non-pathogenic microorganism, the mixture so obtained and a method and use of said mixture for validating the sanitizing of a mill and byproducts obtained therefrom; and an improved method for tempering grain and controlling a population of pathogenic and/or non-pathogenic microorganisms.

Cross Reference to a Related Application

[0001] The present invention claims the priority to US Provisional Application No. 62/924,447 filed October 22, 2019, the entire content of which being incorporated herein by reference in its entirety.

Field of the invention

[0001-a] The present invention relates to a method for preparing a mixture of grain provided with a stable and determined population of at least one pathogenic or non- pathogenic microorganism, the mixture so obtained, and a method and a use of said mixture for validating the sanitizing of a mill and by-products obtained therefrom. Also, the present invention relates to an improved method for tempering grain and controlling pathogenic and/or non-pathogenic microorganisms susceptible to be present in and/or on said grain.

Description of the prior art

[0002] Mills, especially flour mills, comprise various parts that may be contaminated with populations of one or more pathogenic and/or non-pathogenic microorganisms, to thereby contaminate grain contacted with said parts and processed therein, and of course by-products obtained therefrom. Therefore, grain and parts of the mills which are in contact with grain and/or by-products obtained therefrom, need to be sanitized before use.

[0003] Applicant’s international application WO2019/075565, published on April 25, 2019, described a method for tempering grain and controlling the population of pathogenic and/or non-pathogenic microorganisms susceptible to be present in and/or on said grain to thereby obtain a tempered grain. The tempering step is eventually carried out before subjecting the tempered grain to a milling step. [0004] This method comprises a step of contacting a tempering composition with the grain to thereby obtain the tempered grain, 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. The at least one oxidizing agent represents 0.01 to 50% by weight of the oxidizing composition.

[0005] Preferably, the at least one oxidizing agent may comprise: 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 d) optionally at least one additive and/or at least one agriculturally acceptable excipient.

[0006] Also, this method according to international application WO2019/075565 may further contribute to sanitize a milling equipment and may further contribute to produce sanitized by-products.

[0007] In spite of the fact international application WO2019/075565 indicates that using a tempered grain treated with a tempering composition which is a mix of tempering water and an oxidizing composition, may contribute to sanitize various parts of a mill (at least susceptible to be in contact with grain and/or by-products), there is no existing methods allowing to validate that a use of said tempered grain will effectively sanitize said parts of an existing commercial or industrial mill, said parts being at least susceptible to be in contact said tempered grain and/or by-products, and produce sanitized by-products ( e.g . flour) without negatively affecting the physical properties of said by-products.

[0008] Also, validation methods conducted at a mill may be difficult to carry out because it is necessary using a mixture of grain and pathogenic and/or non-pathogenic microorganisms (present in and on the grain). However, mixtures of grain and pathogenic and/or non-pathogenic microorganisms may not be stable over time ( i.e . the load of pathogenic and/or non-pathogenic microorganisms may vary over time). Also, variations of the load of pathogenic and non-pathogenic microorganisms will affect validation results.

[0009] Thus, to carry out a validation method conducted at a mill and by-products obtained therefrom, it is necessary to have a mixture of grain and a population of at least one pathogenic or non-pathogenic microorganisms, where the population of the at least one pathogenic and/or non-pathogenic microorganisms determined and stable over time. However, obtaining such a mixture does not seem to be possible before the present invention was made.

[0010] Therefore, there is a very strong need for a method allowing to obtain a mixture of grain and at least one pathogenic or non-pathogenic microorganism where the population of said at least one pathogenic or non-pathogenic microorganism is determined and stable over time, and of course there is a strong need for such a mixture of grain and at least one pathogenic or non-pathogenic microorganism where the population of said at least one pathogenic or non-pathogenic microorganism is determined and stable over time.

[0011] Also, there is a strong need for an efficient method allowing to carry out a validation method concerning a sanitizing of parts of a mill which are at least susceptible to be in contact with grain and/or by-products obtained therefrom; and a validation method concerning the sanitizing of said by-products.

[0012] The Applicant has surprisingly discovered a method allowing to obtain a mixture of grain and at least one pathogenic or non-pathogenic microorganism where the population of said at least one pathogenic or non-pathogenic microorganism is determined and stable over time.

[0013] Also, the Applicant has surprisingly discovered a mixture of grain and at least one pathogenic and/or non-pathogenic microorganisms having a determined population of said at least one pathogenic and non-pathogenic microorganisms, said population being stable over time. Said mixture is preferably useful for carrying out a validation method for the sanitizing of parts of a mill and by-products obtained from said mill. [0014] Also, the Applicant has surprisingly discovered a validation method allowing to validate the sanitizing of a mill and of by-products obtained therefrom. Also, the Applicant has surprisingly discovered that the validation method does not significatively altered properties and functionalities of the by-products ( e.g . flour).

[0015] Also, the Applicant has surprisingly discovered an improved method for tempering grain and controlling a population of pathogenic and/or non-pathogenic microorganisms present in and/or on said grain.

[0016] Also, the Applicant has discovered a method to mix large quantities of grain and similar food products with pathogenic and non-pathogenic microorganisms, which is needed to conduct industrial-scale validations, including at mills.

Summary of the invention

[0017] One embodiment of the present invention relates to a method for providing a mixture comprising grain and a determined and stable population of at least one pathogenic or non-pathogenic microorganism, wherein said method comprises the steps of:

A) providing an amount of the grain, said grain containing a natural amount of water,

B) providing an amount of an aqueous medium containing an initial population of the at least one pathogenic or non-pathogenic microorganism,

C) contacting the amount of the grain of step A) with the amount of the aqueous medium containing the initial population of the at least one pathogenic or non-pathogenic microorganism of step B), to obtain a mixture;

D) optionally allowing the initial population of the at least one pathogenic or non-pathogenic microorganism contained in the mixture of step C) to grow to thereby obtain a mixture of said amount of the grain, the aqueous medium and a determined population of the at least one pathogenic or non-pathogenic microorganism, E) drying the mixture obtained in step C) or D) in a dryer at a temperature varying from 15 °C to 60 °C to adjust the amount of water contained in the grain to obtain a mixture of the grain with a water content close to its natural content and the determined population of the at least one pathogenic or non-pathogenic microorganism; and

F) recovering and storing the mixture obtained from step E), the mixture obtained from step E) having a determined and stable population of the at least one pathogenic or non-pathogenic microorganism over time.

[0018] Another embodiment of the present invention relates to a mixture comprising grain and a determined population of at least one pathogenic or non-pathogenic microorganism, said mixture having a stable population of said at least one pathogenic or non-pathogenic microorganism and obtained from the method defined hereinabove. Preferably, the at least one pathogenic or non-pathogenic microorganism is present in and/or on said grain.

[0019] Another embodiment of the present invention relates to a validation method for validating the sanitizing a mill and by-products obtained therefrom, said method comprising the steps of: a) providing a mixture of grain and a determined population of at least one pathogenic or non-pathogenic microorganism defined hereinabove, b) tempering the mixture of step a) with a tempering composition comprising: tempering-water, 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 c) milling the tempered grain obtained from step b) into a mill to obtain at least one by-product showing a reduction of the population of pathogenic and/or non-pathogenic microorganisms of at least 2 log, preferably at least 3 log, more preferably about 4 log, no significant regrowth of the population of pathogenic and/or non-pathogenic microorganisms up to at least two-weeks after the tempering of the grain, and no significant alteration of at least one of physical properties and functionalities of the at least one by-product.

[0020] Another embodiment of the invention relates to a use of a mixture of grain and a determined and stable population of at least one pathogenic or non-pathogenic microorganism as defined hereinabove, for validation of the sanitizing of a mill and byproducts obtained therefrom.

[0021] Another embodiment of the invention relates to an improved method for tempering grain and controlling a population of pathogenic and/or non-pathogenic microorganisms present in and/or on said grain, tempered grain so obtained and at least one of by-product to be obtained from said tempered grain, said at least one byproduct showing a reduction of the population of pathogenic and/or non-pathogenic microorganisms of at least 2 log, preferably at least 3 log, more preferably about 4 log, no significant regrowth of the population of pathogenic and/or non-pathogenic microorganisms up to at least two-weeks after the tempering of the grain, and no significant alteration of at least one of physical properties and functionalities of the at least one by-product; wherein said method comprises: a) contacting the grain (eventually in admixture with at least one pathogenic or non-pathogenic microorganism) with a first tempering composition, said first tempering composition comprising 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, to obtain a mixture of said grain and the tempering composition; b) transferring the mixture obtained from step a) into a first tempering capacity and allowing said mixture to temper the grain for a first tempering time, and provide first tempered grain; c) recovering the first tempered grain obtained from step b); d) contacting the first tempered grain of step c) with an amount of a second tempering composition, said second tempering composition comprising water, to obtain a second mixture; e) transferring the second mixture obtained from step d) into a second tempering capacity and allowing said mixture to temper the grain of the first tempered grain for a second tempering time, to provide second tempered grain; and f) recovering the second tempered grain from the second tempering capacity.

Detailed description of the invention

[0022] As mentioned above, one of the embodiments of the invention relates to a method for providing a mixture comprising grain and a determined population of at least one pathogenic or non-pathogenic microorganism, said method comprising the steps of:

A) providing an amount of the grain, said grain containing a natural amount of water, preferably a moisture content varying from 8 wt.% to 14 wt.%,

B) providing an amount of an aqueous medium containing an initial population of the at least one pathogenic or non-pathogenic microorganism,

C) contacting the amount of the grain of step A) with the amount of the aqueous medium containing the initial population of the at least one pathogenic or non-pathogenic microorganism of step B), to obtain a mixture;

D) optionally allowing the initial population of the at least one pathogenic or non-pathogenic microorganism contained in the mixture of step C) to grow to thereby obtain a mixture of said amount of the grain, the aqueous medium and a determined population of the at least one pathogenic or non-pathogenic microorganism, E) drying the mixture obtained in step C) or D) in a dryer at a temperature varying from 15 °C to 60 °C to adjust the amount of water contained in the grain to obtain a mixture of the grain with a water content close of its natural content and the determined population of the at least one pathogenic or non-pathogenic microorganism; and

F) recovering and storing the mixture obtained from step E), the mixture obtained from step E) having a determined and stable population of the at least one pathogenic or non-pathogenic microorganism over time.

[0023] The Applicant has surprisingly discovered that when returning the water content of grain close to its natural water content, the mixture of the grain and the at least one pathogenic and non-pathogenic microorganism shows a stable amount of said at least one pathogenic or non-pathogenic microorganism over time. Preferably, the at least one pathogenic or non-pathogenic microorganism is present in and/or on the grain.

[0024] Another embodiment of the invention relates to the method defined hereinabove, wherein step C) is carried out in a mixer at ambient temperature, preferably at a temperature varying from 15 °C to 40 °C, the amount of the aqueous medium containing the initial population of the at least one pathogenic or non- pathogenic microorganism being contacted with the grain and mechanically mixed with the grain. Preferably, the contact of the aqueous medium containing the initial population of the at least one pathogenic or non-pathogenic microorganism with the grain may be carried out by any appropriate means well known to persons skilled in the art such as pumping, fumigating, spraying, misting or vaporizing. More preferably, the aqueous medium is at least sprayed on the grain, and optionally in a mixer such as a drum mixer. Much more preferably, the aqueous medium is delivered into a spray mechanism by a pump and applied to the grain as it is tumbled in a drum]

[0025] Another embodiment of the invention relates to the method defined hereinabove, wherein step C) is carried out in a mixer at ambient temperature, preferably a temperature varying from 15°C to 40°C. A flow of an aqueous medium containing the initial population of the at least one pathogenic or non-pathogenic microorganism being continuously contacted with a flow of the grain. Preferably, the contact may be carried out by any appropriate means well known to persons skilled in the art. More preferably, the flow of the aqueous medium is sprayed continuously on the grain. More preferably, the aqueous medium is delivered into a spray mechanism by a pump and applied to the grain as it is tumbled in a drum.

[0026] Another embodiment of the invention relates to the method defined hereinabove, wherein step D) is carried out in a capacity, such as a reservoir, a silo, a tank, etc., for a period of at least 1 hours at ambient temperature, preferably a temperature varying from 15°C to 40°C.

[0027] Another embodiment of the invention relates to the method defined hereinabove, wherein step E) is carried out in a continuous dryer.

[0028] Preferably, the continuous dryer is continuous dryer is a vibratory fluidized bed dryer.

[0029] Another embodiment of the invention relates to the method defined hereinabove, wherein, wherein step E) is carried at a temperature of about 40°C.

[0030] Another embodiment of the invention relates to the method defined hereinabove, wherein the at least one pathogenic or non-pathogenic microorganism is selected from the group consisting of E. faecium, Salmonella, E. coli and - Listeria.

[0031] Another embodiment of the invention relates to the method defined hereinabove, wherein the at least one pathogenic or non-pathogenic microorganism is selected from the group consisting of E. faecium.

[0032] Another embodiment of the invention relates to the method defined hereinabove, wherein the grain are any kind of grains that can be milled. Preferably, grains include seeds that can be milled. More preferably, grains are selected from the group consisting of rice, corn, wheat and raw crop grains. Much more preferably, grain is wheat such as hard or soft wheat.

[0033] Another embodiment of the invention relates to a mixture comprising grain and a population of at least one pathogenic or non-pathogenic microorganism, said mixture having a determined and stable population of the at least one pathogenic or non-pathogenic microorganism, and being obtained from the method defined hereinabove.

[0034] Another embodiment of the invention relates to the mixture defined hereinabove, wherein the population of the at least one pathogenic and non-pathogenic microorganism varies from 3 log CFU/g to 8 log CFU/g], and the natural water content of the grain varies from 8wt.% to 14wt.% moisture content.

[0035] Another embodiment of the invention relates to the mixture defined hereinabove, wherein the population of the at least one pathogenic or non-pathogenic microorganism is stable for a period of at least 2 weeks after step E) when stored at a temperature varying from 4.0°C to 40°C.

[0036] According to another embodiment, the invention relates to the mixture defined hereinabove, wherein the population of the at least one pathogenic or non- pathogenic microorganism is stable for a period of at least 2 weeks after step E) when stored at a temperature varying from 4.0 °C to 40 °C and relative humidities varying from 30-70%.

[0037] Another embodiment of the invention relates to the mixture defined hereinabove, wherein the at least one pathogenic or non-pathogenic microorganism is selected from the group consisting of E. faecium, Salmonella, E. coli. And Listeria.

[0038] Another embodiment of the invention relates to the mixture defined hereinabove, wherein the at least one pathogenic or non-pathogenic microorganism is selected from the group consisting of E. faecium.

[0039] Another embodiment of the invention relates to the mixture defined hereinabove, wherein the grain refer to all grains that can be milled. Preferably, grains include seeds that can be milled. More preferably, grains are selected from the group consisting of rice, corn, wheat and raw crop grains. Much more preferably, grain is wheat such as hard or soft wheat.

[0040] Another embodiment of the invention relates to a use of a mixture of grain and a determined population of at least one pathogenic or non-pathogenic microorganism, as defined above, for validating the sanitizing a mill and by-products obtained therefrom.

[0041] Another embodiment of the invention relates to the mixture defined hereinabove, wherein the pathogenic and non-pathogenic microorganism are selected from the group consisting of E. faecium, Salmonella, E. coli. and Listeria.

[0042] Another embodiment of the invention relates to the mixture defined hereinabove, wherein the pathogenic and non-pathogenic microorganisms are selected from the group consisting of E. faecium.

[0043] Another embodiment of the invention relates to the mixture defined hereinabove, wherein grain refer to all grains that can be milled. Preferably, grains include seeds that can be milled. More preferably, grains are selected from the group consisting of rice, corn, wheat and raw crop grains. Much more preferably, grain is wheat such as hard or soft wheat.

[0044] Another embodiment of the invention relates to the mixture defined hereinabove, wherein the at least one by-product is a flour.

[0045] Another embodiment of the invention relates to a validation method for validating the sanitizing a mill and by-products obtained therefrom, said method comprising the steps of: a) providing a mixture of grain and a determined population of at least one pathogenic or non-pathogenic microorganism as defined hereinabove, b) tempering the mixture of step a) with a tempering composition comprising: tempering-water, an oxidizing composition which 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, to obtain a tempered grain; and c) milling the tempered grain obtained from step b) into a mill to obtain at least one by-product showing a reduction of the population of pathogenic or non-pathogenic microorganism of at least 2 log, preferably at least 3 log, more preferably about 4 log, no significant regrowth of the population of pathogenic and/or non-pathogenic microorganisms up to at least two- weeks after the tempering of the grain, and no significant alteration of at least one of physical properties and functionalities of the at least one byproduct.

[0046] Another embodiment of the invention relates to the validation method as defined hereinabove, wherein the tempering of the mixture of step 1) comprises at least one tempering step.

[0047] Another embodiment of the invention relates to a validation method for validating the sanitizing of a mill and by-products obtained therefrom, said method comprising the steps of: a) providing a mixture of grain and a determined population of at least one pathogenic or non-pathogenic microorganism defined hereinabove, b) tempering the mixture of step a), said tempering comprising the steps of b1) contacting the mixture of step a) with a first tempering composition, said first tempering composition comprising:

- tempering-water,

- 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, to obtain a mixture of said grain and the tempering composition; b2) transferring the mixture obtained from step b1) into a first tempering capacity and allowing said mixture to temper the grain for a first tempering time, and provide first tempered grain; b3) recovering the first tempered grain obtained from step b2); b4) contacting the first tempered grain of step b3) with an amount of a second tempering composition, said second tempering composition comprising water, to obtain a second mixture; b5) transferring the second mixture obtained from step b4) into a second tempering capacity and allowing said mixture to temper grain of the first tempered grain for a second tempering time, to provide second tempered grain; and b6) recovering the second tempered grain from the second tempering capacity; and c) milling the tempered grain obtained from step b6) into a mill to obtain at least one by-product showing a reduction of the population of pathogenic and/or non-pathogenic microorganisms of at least 2 log, preferably at least 3 log, more preferably about 4 log, no significant regrowth of the population of pathogenic and/or non-pathogenic microorganisms up to at least two-weeks after the tempering of the grain, and no significant alteration of at least one of physical properties and functionalities of the at least one by-product.

[0048] Another embodiment of the invention relates to any one of the validation methods defined hereinabove, wherein the mixture of the grain and the determined population of the at least one pathogenic or non-pathogenic microorganism is as defined hereinabove as an embodiment of the invention, is obtained according to the method defined hereinabove as an embodiment of the inventio, or both.

[0049] Another embodiment of the invention relates to the validation method defined hereinabove, wherein the at least one oxidizing agent represents 0.01 to 50% by weight of the oxidizing composition.

[0050] Another embodiment of the invention relates to the validation method defined hereinabove, wherein the oxidizing composition and the tempering-water of the first tempering composition are a weight ratio varying from 4:16 to 2:78. [0051] Another embodiment of the invention relates to the validation method defined hereinabove, wherein the weight ratio varies from 3:16 to 2:78.

[0052] Another embodiment of the invention relates to the validation method defined hereinabove, wherein the first tempering composition is applied to the grain at a rate varying from 20 to 100 liters of the tempering composition per ton of grain.

[0053] Another embodiment of the invention relates to the validation method defined hereinabove, wherein the first tempering time varies from 1 hours to 24 hours.

[0054] Another embodiment of the invention relates to the validation method defined hereinabove, wherein the first tempering time is about 16 hours.

[0055] Another embodiment of the invention relates to the validation method defined hereinabove, wherein the second tempering time varies from 1 to 24 hours.

[0056] Another embodiment of the invention relates to the validation method defined hereinabove, wherein the second tempering time is about 4 hours.

[0057] Another embodiment of the invention relates to the validation method defined hereinabove, wherein the first tempering composition is contacted with the grain by any appropriate means, such as pumping, fumigating, spraying, misting or vaporizing on the grain, optionally in a mixer such as a drum mixer. Spraying is particularly preferred.

[0058] Another embodiment of the invention relates to the validation method defined hereinabove, wherein the second tempering composition is contacted with the first tempered grain of step b4) by any appropriate means such as pumping, fumigating, spraying, misting or vaporizing on the grain, optionally in a mixer such as a drum mixer. Spraying is particularly preferred.

[0059] Another embodiment of the invention relates to the validation method defined hereinabove, wherein the first tempering capacity is a first tempering tank.

[0060] Another embodiment of the invention relates to the validation method defined hereinabove, wherein the second tempering capacity is a second tempering tank. [0061] Another embodiment of the invention relates to the validation method defined hereinabove, wherein said oxidizing composition comprises the at least one oxidizing agent in liquid form or solid form, or a precursor thereof in liquid or solid form.

[0062] Another embodiment of the invention relates to the validation method defined hereinabove, wherein the at least one oxidizing agent comprises: a) liquid peracid and/or in-situ generated peracid; b) liquid hydrogen peroxide and/or hydrogen peroxide released from a hydrogen peroxide precursor, and/or c) other liquid oxidizers and/or powder oxidizers generating iodine, chlorine, bromine and/or chlorine dioxide.

[0063] Another embodiment of the invention relates to a validation method defined hereinabove, 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.

[0064] Another embodiment of the invention relates to a validation method defined hereinabove, wherein the at least one oxidizing agent is an oxidizing composition obtained by admixture of an aqueous solution of peracetic acid obtained from the reaction of acetic acid with hydrogen peroxide; with an aqueous solution of hydrogen peroxide, said oxidizing composition comprising:

• peracetic acid in an amount varying from 0.1% to 5% by weight of the oxidizing composition,

• hydrogen peroxide in an amount varying from 0.1% to 20% by weight of the oxidizing composition, and

• water in an amount varying from 75% to 99.8% by weight of the oxidizing composition. [0065] Another embodiment of the invention relates to the validation method defined hereinabove, wherein the oxidizing composition has the following formulation:

[0066] Another embodiment of the invention relates to the validation method defined hereinabove, wherein the pathogenic and/or non-pathogenic mircoorganismsare selected from the group consisting of E. faecium, Salmonella and E. coli. and Listeria.

[0067] Another embodiment of the invention relates to the validation method defined hereinabove, wherein the pathogenic and/or non-pathogenic microorganisms are selected from the group consisting of E. faecium.

[0068] Another embodiment of the invention relates to the validation method defined hereinabove, wherein grain refer to all type of grains that can be milled mixture of grain and a determined population of at least one pathogenic or non-pathogenic microorganism defined hereinabove. [0069] Another embodiment of the invention relates to the validation method defined hereinabove, wherein the at least one by-product is a flour, optionally a flour obtained by milling of the second tempered grain.

[0070] Another embodiment of the invention relates to an improved method for tempering grain and controlling a population of pathogenic and/or non-pathogenic microorganisms present in and/or on said grain, tempered grain so obtained and at least one of by-product obtained from said tempered grain, said at least one by-product showing a reduction of the population of pathogenic and/or non-pathogenic microorganisms of at least 2 log, preferably at least 3 log , more preferably about 4 log, no significant regrowth of the population of pathogenic and/or non-pathogenic microorganisms up to at least two-weeks after the tempering of the grain, and no significant alteration of at least one of physical properties and functionalities of the at least one by-product; wherein said method comprises: a) contacting the grain with a first tempering composition, said first tempering composition comprising 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, to obtain a mixture of said grain and the tempering composition; b) transferring the mixture obtained from step a) into a first tempering capacity and allowing said mixture to temper the grain for a first tempering time, and provide first tempered grain; c) recovering the first tempered grain obtained from step b); d) contacting the first tempered grain of step c) with an amount of a second tempering composition, said second tempering composition comprising water, to obtain a second mixture; e) transferring the second mixture obtained from step d) into a second tempering capacity and allowing said mixture to temper grain of the first tempered grain for a second tempering time, to provide second tempered grain; f) recovering the second tempered grain from the second tempering capacity.

[0071] Another embodiment of the invention relates to the improved method defined hereinabove, wherein sanitized by-products can be obtained by mechanically transforming ( e.g . milling) the second tempered grain obtained from step f) to provide at least one by-product thereof.

[0072] Another embodiment of the invention relates to the improved method defined hereinabove, wherein the at least one oxidizing agent represents 0.01 to 50% by weight of the oxidizing composition.

[0073] Another embodiment of the invention relates to the improved method defined hereinabove, wherein the oxidizing composition and the tempering-water of the first tempering composition are a weight ratio varying from 4:16 to 2:78.

[0074] Another embodiment of the invention relates to the improved method defined hereinabove, wherein the weight ratio varies from 3:16 to 2:78.

[0075] Another embodiment of the invention relates to the improved method defined hereinabove, wherein the first tempering composition is applied to the grain at a rate varying from 20 to 100 liters of the tempering composition per ton of grain.

[0076] Another embodiment of the invention relates to the improved method defined hereinabove, wherein the first tempering time varies from 1 hours to 24 hours.

[0077] Another embodiment of the invention relates to the improved method defined hereinabove, wherein the first tempering time is about 16 hours.

[0078] Another embodiment of the invention relates to the improved method defined hereinabove, wherein the second tempering time varies from 1 to 24 hours.

[0079] Another embodiment of the invention relates to the improved method defined hereinabove, wherein the second tempering time is about 4 hours, more preferably about 1 hours. [0080] Another embodiment of the invention relates to the improved method defined hereinabove, wherein the first tempering composition is contacted with the grain by any appropriate means, such as pumping, fumigating, spraying, misting or vaporizing on the grain. More preferably, the first tempering composition is sprayed on the grain, and optionally in a mixer such as a drum mixer.

[0081] Another embodiment of the invention relates to the improved method defined hereinabove, wherein the second tempering composition is contacted with the first tempered grain of step c) by any appropriate means, such as by pumping, fumigating, spraying, misting or vaporizing on the grain. More preferably, the second tempering composition is sprayed on the grain, and optionally in a mixer such as a drum mixer.

[0082] Another embodiment of the invention relates to the improved method defined hereinabove, wherein the first tempering capacity is a first tempering tank.

[0083] Another embodiment of the invention relates to the improved method defined hereinabove, wherein the second tempering capacity is a second tempering tank.

[0084] Another embodiment of the invention relates to the improved method defined hereinabove, wherein said oxidizing composition comprises the at least one oxidizing agent in liquid form or solid form, or a precursor thereof in liquid or solid form.

[0085] Another embodiment of the invention relates to the improved method defined hereinabove, wherein the at least one oxidizing agent comprises: a) liquid peracid and/or in-situ generated peracid; b) liquid hydrogen peroxide and/or hydrogen peroxide released from a hydrogen peroxide precursor, and/or c) other liquid oxidizers and/or powder oxidizers generating iodine, chlorine, bromine and/or chlorine dioxide.

[0086] Another embodiment of the invention relates to the improved method defined hereinabove, 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.

[0087] Another embodiment of the invention relates to the improved method defined hereinabove, wherein the at least one oxidizing agent is an oxidizing composition obtained by admixture of an aqueous solution of peracetic acid obtained from the reaction of acetic acid with hydrogen peroxide; with an aqueous solution of hydrogen peroxide, said oxidizing composition comprising:

• peracetic acid in an amount varying from 0.1% to 5% by weight of the oxidizing composition,

• hydrogen peroxide in an amount varying from 0.1% to 20% by weight of the oxidizing composition, and

• water in an amount varying from 75% to 99.8% by weight of the oxidizing composition.

[0088] Another embodiment of the invention relates to the improved method defined hereinabove, wherein the oxidizing composition has the following formulation: [0089] Another embodiment of the invention relates to the improved method defined hereinabove, wherein the pathogenic and/or non-pathogenic microorganisms are selected from the group consisting of E. faecium, Salmonella, E. coli. and Listeria.

[0090] Another embodiment of the invention relates to the improved method defined hereinabove, wherein the pathogenic and/or non-pathogenic microorganisms are selected from the group consisting of E. faecium.

[0091] Another embodiment of the invention relates to the improved method defined hereinabove, wherein grain refer to all kind of grains that can be milled mixture of grain and a determined population of at least one pathogenic or non-pathogenic microorganism defined hereinabove.

[0092] Another embodiment of the invention relates to the improved method defined hereinabove, wherein the at least one by-product is a flour, optionally a flour obtained by milling of the second tempered grain.

[0093] According to another embodiment of the invention, a measure of the determined amount of the at least one pathogenic and/or non-pathogenic microorganism is obtained by any analytical methods well known to persons skilled in the art. Preferably, examples of such analytical methods include the procedures described in the FDA Bacteriological Analytical Manual (BAM; Andrews and Hammack, 2003 which is incorporated herein by reference.

[0094] According to another embodiment of the invention, the expression «a stable population of the at least one pathogenic or non-pathogenic microorganism» means the population that does not increase or decrease by more than 0.50 log CFU/g over a period of time of at least 2 weeks.

[0095] According to another embodiment of the invention, the expression «no significant regrowth of the population of the at least one pathogenic or non-pathogenic microorganism» means that the population did not increase by more than 0.5 log CFU/g, more preferably over a period of time of at least 2 weeks. [0096] According to another embodiment of the invention, the expression «no significant alteration of physical properties of by-products» means that no significant changes in moisture, protein, ash, pH of the resultant flour.

[0097] According to another embodiment, the expression » no significant alteration of functionalities of by-products» refer to bread baking qualities, such sensory (bake score), ability of the dough to rise and maintain shape (bake strength), water absorption, stability of the flour under mixing, dough viscosity, peak mixing time to arrive at desired water/gluten ratio, etc.].

Brief description of the drawings

[0098] The following description will be better understood with reference to the following drawings illustration particularly preferred embodiments of the invention

• Figure 1 represents concerning example 1 , a graph of the variation of the temperature and couple (Nm) versus time for a sample.

• Figure 2 represents a raised dough obtained from a flour according to the present example and ready to be cooked.

• Figure 3 represents a photograph of a slice of a bread obtained after cooking the dough of Figure 2.

• Figure 4 represents, at 1.5% moisture, a log reduction of E. faecium in wheat and a log reduction of E. faecium on flour.

• Figure 5 represents, at 3.0% moisture, a log reduction of E. faecium in wheat and a log reduction of E. faecium on flour.

• Figure 6 represents, at 4.5% moisture, a log reduction of E. faecium in wheat and a log reduction of E. faecium on flour.

[0099] There will be provided hereinafter some examples illustrating particularly preferred and non-limiting embodiments of the invention

[0100] In the following examples, use was made of an industrial apparatus or a combination of industrial apparatuses. When applying an inoculum and drying the resulting mixture, an apparatus well known to persons skilled in the art under the commercial name of NEO-PURE CONTINUOUS FOOD SYSTEM was used. It comprised a NEO PURE APPLICATOR device (i.e. defining an application section) and a NEO PUR dryer (i.e. defining a fluidized bed dryer section). Both sections were connected through a Cablevey conveyor. It was or they were controlled by a central programmable logic controller (PLC) system. Of course, when contacting the inoculated grain with a tempering composition, the step concerning the fluidized bed dryer section was merely omitted.

[0101] The application section was provided with a mixing unit comprising

- an inlet (i.e. a hopper having a 300kg capacity),

- an outlet (i.e. an exit chute),

- a rotary valve positioned at the bottom of the hopper to control the flow rate of grain entering in the application section, and then the subsequent fluidized bed dryer section,

- a rotary drum (a 7.5 x 3.5 foot diameter stainless steel drum provided with folded baffles inside, which rotates and mixes the grain for a thorough coverage),

- a conduit in fluid communication between the hopper and one end of the rotary drum, at least one part of the conduit defining an atomizer section;

- one peristaltic pump adapted to transfer in a controlled manner the fluid product to a nozzle and form a spray of the liquid product contacting the grain moving in the conduit.

[0102] The nozzle was covered with a protective mesh screen, and spinning radially (in the atomizer section), to spray the fluid product onto the grain to maximize coverage. The rotary valve was consisting of pockets which rotate and are filled with grain falling by gravity from the hopper. The rotary valve was receiving the grain from the hopper and was feeding them into a cylinder that has a cone inside. After falling onto the cone, the grain was spread and allowed to fall like a «cylindric curtain» shape into the atomizer section

[0103] Preferably, the fluid product was either a composition comprising an inoculum, or a tempering composition. When the fluid product was a tempering composition, three peristaltic pumps were provided to inject reactants into a further static mixer, and then form said tempering composition which exits of the static mixer and then shortly before reaching and exiting the nozzle, forms a spray of the tempering composition in the atomizer section.

[0104] The fluidized bed dryer section had the following particulars.

• The dimension of the dryer was 24x4.5 feet and was consisting of three 6-foot drying sections and one 6-foot cooling section. The dryer bed platform was oscillating forward, which gave the grain movement. The bed platform was made of wedge wires to allow the hot air to circulate upward. The dryer was provided with an inlet end and an outlet end.

• Hot and cool air was entering the dryer from the bottom of the dryer and was exhausted out from the top. Air velocity and vibration frequency were fixed at certain values and were not be changeable by operators. Hot and cool air were filtered through MERV 13 filters.

• Grain feed rate was dependent on the feed rate of the rotary valve prior to the applicator, and was set to 3 metric tons/h. Grain bed depth and dwell time in the dryer were dependent on the feed rate and static weirs placed throughout each of the three heated sections. The dwell time in the dryer was 7 minutes. The weirs were only raised at the end of process to let grain flow freely and exit the dryer. Prior to lifting the weirs, the dryer bed depth was held for an additional 7 minutes to assure all the grain was dried to the correct level.

• Temperature was measured by Resistance Temperature Detectors (RTD) located at the end of drying section, located one third of the width from each side. This location was selected after temperature mapping indicated this was the coldest spot in the dryer. Air temperature was measured in 1 -second intervals prior to the grain bed and transmitted to the programmable Logic Controller (PLC). The RTD with the lowest temperature controls the process. Concerning inoculation, a minimum temperature is not critical in the drying section because the temperature contributes to remove an excess of moisture. However, when treating a food to kill bacterial, if the temperature felt below a critical Temperature to Be Determined (TBD) the system was shut down. Indeed, in such a case, if the temperature falls below a critical minimum temperature, the efficacy may be compromised. Consequently, in such a case, when at a critical control point (CCP)), the temperature felt below the critical temperature, the system will be shut down.

• When necessary, the outlet of the application section was connected to the inlet of the dryer by a Cablevey Conveyor system.

[0105] The protocol involved for the following examples (except otherwise indicated) was as follows:

• Concerning the application section:

- The grain was fed into the atomizer through rotary valve at 3 metric tons/h flow rate.

- The grain was then sprayed with the fluid product while passing through the atomizer at determined L/metric ton application rate and then was entering the rotary drum. Treated grain get mixed inside the rotary drum that rotates horizontally with a residence time of 60 seconds.

- The treaded grain was exiting the rotary drum.

• Concerning both the application section and the fluidized bed dryer section:

- The above parameters were repeated concerning the application section.

- The treated grain was exiting the rotary drum, entering the inlet hopper of the Cablevey conveyor which is located right under drum exit chute. The grain then get conveyed to the top of the dryer and was entering the dryer through its inlet hopper.

- There were 3 static weirs inside the dryer that ensure that the grain resided in the dryer for 7 min before they enter the cooling section. The grain remained in the cooling section for 2 min and then was allowed to exit the dryer. A weir at the end of cooling section was ensuring that the grain was remaining in the cooling section for about 2 min. Example 1 Efficacy of treating wheat inoculated with Enterococcus faecium NRRL B- 2354 a surrogate for Salmonella and Escherichia coli with L/t Neo-Temper during tempering method.

1) Preparation of a mixture of wheat grain and E. faecium NRLL B-2354

[0106] Five hundred kg of wheat was inoculated with overnight culture of E. faecium NRRL B-2354 at a rate of 6% (60 ml inoculum per kg) followed by drying at 105 °F (40 °C) in the continuous dryer (fluid bed dryer) defined above. The moisture content of the inoculated wheat was 12.15%. The moisture content before inoculation was 12.07%. The load of bacteria was 6.73 ± 0.03 log CFU/g on the date of enumeration.

[0107] A single colony grown from a frozen stock of E. faecium was used to inoculate aqueous medium. Successive passages of aqueous medium were conducted to achieve the needed volume of inoculum for 500 kg of wheat. Inoculum was delivered to a spray mechanism by a pump and applied to the wheat as it was tumbled in a mixer before being conveyed into the fluid bed dryer.

[0108] The inoculated wheat so obtained was then shipped out to an industrial mill. The inoculated wheat showed having a stable population of E. Faecium for at least two weeks at ambiant temperature (i.e. about 22°C).

2) First tempering of the inoculated wheat obtained from step 1) at the industrial mill (about 72 hours after the inoculation).

[0109] A fresh tempering composition was prepared on-site prior to treatment and its strength was confirmed to be within manufacturer’s recommended range using Masters kits that apply Photometric Measuring Technology. The equipment necessary to apply tempering composition on the inoculated wheat was also adjusted and calibrated to ensure accuracy of application rate.

[0110] The tempering composition was prepared by mixing 26.6 liters of water (i.e. tempering water) with 6.90 liters of a commercial product known under the tradename Neo-Temper. The Neo-Temper comprises:

[0111] Then, the tempering composition was contacted with inoculated wheat at a rate of 33.5 L/ton (i.e. 16.75 L /500kg) by spraying on the inoculated wheat in a mixer and conveyed into a first tempering silo and stored therein for about 16 hours.

3) Second tempering of the tempered wheat obtained from step 2)

[0112] The tempered wheat obtained from step 2) was recovered from the first tempering silo, and then sprayed with more water at a rate of 26.6 L/ton in a mixer, and then conveyed into a second tempering silo and stored therein for about 4 hours.

4) Milling

[0113] The tempered wheat obtained from step 3) was recovered from the second tempering silo, and then milled in an industrial roller mill at a flow rate of 3 t/hour. A fine wheat flour was produced. 5) Sampling

[0114] Five samples (approx. 1 kg each) were taken from fine flour produced at the end of step 4 (i.e. end of the milling line) and from untreated control wheat to conduct microbiological analysis. Also, five 1 kg samples were also taken from the fine flour to conduct Functionality analysis.

6) Micro analysis

[0115] Enumeration of E. faecium was done following the procedure described in the FDA Bacteriological Analytical Manual (BAM) (Andrews and Hammack, 2003). Briefly, each 45-g sample of wheat or flour was mixed with 90 (1:2 w/v) or 180 (1:4 w/v) ml of buffered peptone water (BPW), respectively, in a sterile stomacher bag, followed by blending for 2 min at 230 RPM in a paddle blender before preparing 10-fold serial dilutions in BPW. Plating was done on Enterococci selective agar (Slanetz & Bartley), followed by incubation at 95 °F (35 °C) for 48 h. Results are reported in log CFU/g. The detection limit was either 2 (0.3 log CFU/g) or 4 CFU/g (0.6 log CFU/g), depending on the dilutions used. Treated flour samples were also plated 2 weeks after treatment to study potential regrowth of Salmonella surrogate.

7) Quality and functionality

[0116] Chemical and rheological evaluation as well as baking quality of the samples was also evaluated and compared to a blank as a reference. Chemical evaluation was performed with near-infrared (Foss NIRSystems 6500) for humidity, protein and ash levels, and falling number (Perten) for amylase activity (AACCI/No. 56-81.03). It is to be noted that protein, moisture, and ash content are the principle parameters measured to determine wheat flour quality. These parameters are commonly quantified using near-infrared (NIR) spectroscopy. NIR is a spectroscopic method that uses the near- infrared region of the electromagnetic spectrum. Spectroscopy allows the composition, physical structure and electronic structure of matter to be investigated. Also, rheological analysis was done through flour analysis on Glutopeak (Perten) at 36 °C, speed 2,750 rpm, 300 seconds, and Mixolab (Chopin) using the standard Chopin S protocol. Finally, baking tests were carried out using a VMI Spiral mixer as per manufacturer recommendations. Results

8) Microbiology results: a) Efficacy post-treatment:

[0117] The results are shown in the following tables I and II, demonstrating that Neo-Temper was able to reduce the population of E. faecium from 6.73 ± 0.03 to 2.84 ± 0.27 LogCFU/g, producing an average of 3.90 log reduction in the flour.

[0118] Such a log reduction in the flour also illustrate that the parts of the mill being in contact with the tempered grain and/or the flour have been sanitized too.

[0119] The inoculation technique used for inoculating 500 kg of wheat proved to be very effective and efficient with standard deviation of as small as 0.03.

[0120] Neo-Temper also was very efficient in reducing population of Salmonella and E. coli surrogate by about 4 log, again with a small standard deviation of 0.27, which shows enhanced coverage achieved on the wheat kernels during tempering.

[0121] This enhanced coverage of Neo-Temper enables it to reach microbial cells harboured in hard-to-reach areas (cracks, crevices, etc.) that are generally called harbourage areas.

Table I Table II b) Regrowth study:

[0122] The results (see table III) show that 2.98 ± 0.12 log CFU/g of E. faecium was recovered from the treated samples stored in ambient temperature 73 °F (23 °C) for 2 weeks. No significant difference was observed in the population of Salmonella and E. coli surrogate recovered at week zero compared to week 2, indicating no regrowth occurred during storage in ambient temperature.

Table III

2. Functionality results:

[0122] Functionality data also suggests that no changes were observed in the parameters tested (moisture, protein, ash, absorption, stability, and weakening, and therefore, functionality of the flour produced post application of Neo-Temper is unaltered and within spec. Results table (see table IV) and photos of the dough and the bread made using Neo-Temper treated flour are illustrated in Figures 1 to 3:

Table IV

[0123] Figure 1 represents a graph of the variation of the temperature and couple (Nm) versus time for a sample (under the following conditions):

[0124] Figure 2 represents a raised dough obtained from a flour according to the present example and ready to be cooked to provide a loaf of bread. [0125] Figure 3 represents a photograph of a slice of a bread obtained from the raised dough of Figure 2.

Example 2

[0126] Analysis of samples that were tempered with Neo-Temper solution to see how it impacts the performance and how effective is the microorganism reduction. More particularly, the Neo-Temper concentration was increased to see if it impacts functionality of flour.

[0127] Testing methods

• Milling Extraction (Flour Yield %)

• MAP (AACC)

• MAP (NIR)

• Farinograph (Constant Flour Weight)

• pH

• Falling Number

• Bread Bake

[0128] Testing methods were carried out as per manufacturer recommendation [0129] Results are contained in the following tables V to X. Table V

1) American Association for Clinical Chemists

2) AACC Method 44-15.02 - Moisture -- Air-Oven Methods: These methods determine moisture content as loss in weight of a sample when heated under specified conditions.

3) AACC Method 08-01 .01 - Ash -- Basic Method: Ash content of flour is defined as the residue remaining after controlled incineration of the flour. It represents the mineral content of the flour.

4) AACC Method 46-15.01 - Crude Protein -- 5-Minute Biuret Method for Wheat and Other Grains : Compounds with peptide linkages produce a blue-violet color with copper ions in alkaline solution. This is named biuret reaction after the chemical compound biuret, first known to produce the color. The color is due to a complex bond formed between cupric ion and four peptide nitrogen atoms, two from each of two adjacent peptide chains.

Table VI

[0130] Near infrared Radiation (NIR): Protein, moisture, and ash content are the principle parameters measured to determine wheat flour quality. These parameters are commonly quantified using near-infrared (NIR) spectroscopy. NIR is a spectroscopic method that uses the near-infrared region of the electromagnetic spectrum. Spectroscopy allows the composition, physical structure and electronic structure of matter to be investigated. Table VII

[0131] A farinograph was the tool used for measuring dough and gluten properties of a flour sample. The results of a farinograph test are used as parameters in formulation to estimate the amount of water required to make dough, evaluate the effects of ingredients on mixing properties, evaluate flour blending requirements, and to check flour uniformity

[0132] It was used as a sensing device that measures the dough’s resistance to mixing during successive stages of its development. At any particular time, dough may be in the process of being stretched, sheared, compressed, or may be in a state of relaxation resulting in the farinograph curve. The curve of the farinograph is a reflection of three processes: absorption of water; dough development; and dough breakdown. In the above table VII:

Absorption is the amount of water required to center the farinograph curve.

Peak Time indicates dough development time, beginning the moment water is added until the dough reaches maximum consistency. Peak time is in minutes and gives an indication of optimum mixing time under standardized conditions.

Stability Time is the difference between arrival time and departure time. It is a good indication of dough strength and is expressed in minutes.

Arrival Time indicates the rate of flour hydration and is expressed in minutes.

Departure Time indicates the time when dough is beginning to break down and is an indication of dough consistency during processing and is expressed in minutes.

Mixing Tolerance Index (MTI) indicates the degree of softening during mixing and is expressed in Brabender units (BU).

[0133] The above table VII corresponds to Figure 1

Q) CT CO CD 00 <

[0134] Microbiological Analysis

Table IX Table X

[0135] Figure 4 represents, at 1.5% moisture, a 2 log reduction of E. faecium in wheat and 1 log reduction in flour. No major difference between Neo-Temper concentrations. [0136] Figure 5 represents at 3.0% moisture: 2 log reduction of E. fecium in wheat. For flour samples, a 2 log reduction was observed from wheat to flour on control sample where moisture was added but no treatment. Neo Temper reduction of E. faecium on flour was approximately 2 log.

[0137] Figure 6 represents, at 4.5% moisture: lowest observed 2 log reduction of E. faecium in wheat. For flour samples, there was a 2 log reduction from wheat to flour on control samples where moisture was added but no treatment. Based on this, NeoTemper reduction of E. faecium on flour was approximately 1 log.

[0138] Conclusion

- There was no significant impact found on flour quality and/or functionalities.

- From inoculated control to control flour there was a 2 log reduction, 1 log from milling and 1 log from “wash off”

- Neo-Temper provided a 2 log reduction of E. faecium on wheat and 1 log reduction on flour.

Example 3

[0139] This example refers to an industrial-scale surrogate validation in a wheat flour mill. More particularly, this example refers to the efficacy of treating soft wheat inoculated with Enterococcus faecium NRRL B-2354 a surrogate for Salmonella and Escherichia coli with 5 and 9 L/t Neo-Temper during tempering process

[0140] The process comprised the following steps:

1) Inoculation:

[0141] Approximately 9,300 kg wheat was inoculated with overnight culture of E. faecium NRRL B-2354 at a rate of 6% (60 ml inoculum per kg) followed by drying at 105 °F (40 °C) in fluidized continuous dryer (i.e. fluid bed dryer) at 3 t/h throughput rate. The moisture content of the inoculated wheat was returned to its original value. The inoculum level was 6.15 ± 0.19. The inoculum was applied in the continuous system, like the other inoculations. Overnight inoculum was applied at 60 L/t rate with the Neo-Pure Applicator and then dried in the Neo-Pure dryer at 105 F. The average fresh wheat moisture content was 10.30%, the average fresh wheat water activity was 0.39, the average inoculated wheat moisture content was 10.70% and the average inoculated water activity was 0.45. The inoculated wheat showed having a stable population of E. Faecium for at least two weeks at ambiant temperature (i.e. about 22°C).

[0142] Then the Inoculated wheat was shipped out to a mill facility to be treated during the tempering process and then milled into flour.

2) Pre-treatment quality control :

[0143] A fresh tempering composition was prepared on-site prior to treatment and its strength was confirmed to be within manufacturer’s recommended range using Masters kits that apply Photometric Measuring Technology. More particularly, Neo-Temper composition was prepared by mixing in-line through the static mixer: (i) Neo-Alpha One (i.e based ingredients comprising in water Peracetic Acid (PAA)), and (ii) Neo-Alpha Two (i.e. based ingredient comprising in water Hydrogen Peroxide). Water was further added to obtain the tempering composition. Concentration of the two ingredients used to prepare fresh Neo-Temper was measured before the run using Masters kits that apply Photometric Measuring Technology and were as followed:

Table XI

3) Treatment:

[0144] The equipment necessary to apply tempering composition on the inoculated wheat was also adjusted and calibrated to ensure accuracy of application rate. The inoculated wheat was sprayed on with final solution of 11.2 L/t including Neo-Temper and water using a Prominent motor-driven metering pump Sigma X model S2Cb. However, half of the wheat was treated with 5 L/t of Neo-Temper (plus 6.2 L/t water) and the remaining half was treated at 9 L/t (plus 2.2 L/t water). Below table shows the Neo-Temper treatment rates, water rates, wheat flow rate, amount of food treated, treatment duration, and tempering duration, for both runs: Table XII

4) Tempering:

[0145] Immediately after treatment, the wheat was conveyed into tempering silos and stored for 6 h.

5) Milling:

[0146] The treated and tempered wheat was then milled in an industrial mill at 27.7 t/hr throughput.

6) Sampling:

[0147] For untreated control samples, 12 samples (1kg each) of inoculated wheat were collected before tempering. During each run 12 samples (1kg each) of fine flour were also collected. The samples were sent for to conduct microbiological analysis at Applicant’s Laboratory. 1 kg samples were also taken to conduct functionality tests by the staff of the milling facilities

7) Micro analysis:

[0148] Enumeration of E. faecium was done at Applicant’s laboratories following the procedure described in the FDA Bacteriological Analytical Manual (BAM) (Andrews and Hammack, 2003). Briefly, each 45-g sample of wheat or flour was mixed with 90 (1:2 w/v) or 180 (1 :4 w/v) ml of buffered peptone water (BPW), respectively, in a sterile stomacher bag, followed by mixing through shaking vigorously 50 times in a 30 cm (1 ft.) arc by hand. Then, the samples were left to stand for 3-5 minutes and shaken vigorously 5 times in a 30 cm arc just before 10-fold serial dilutions in BPW were prepared. Plating was done on Enterococci selective agar (Slanetz & Bartley), followed by incubation at 95 °F (35 °C) for 48 h. Results are reported in log CFU/g. The detection limit was either 2 CFU/g (0.3 log CFU/g) or 4 CFU/g (0.6 log CFU/g), depending on the dilutions used.

8) Microbiological Analysis Results:

[0149] The results are shown in the following tables demonstrate that Neo-Temper was able to reduce the population of E. faecium from 6.15 ± 0.19 to 3.54 ± 0.22 and 3.06 ± 0.3 LogCFU/g at 5 and 9 L/t of Neo-Temper, respectively. This indicates that Neo-temper was able to produce 2.61 and 3.09 log reduction on E. faecium, a surrogate of Salmonella and E. coli, respectively. The inoculation technique used for inoculating approx. 9,300 kg of wheat proved to be very effective and efficient with standard deviation of as small as 0.19. Neo-Temper also was effective in reducing population of Salmonella and E. coli surrogate by about 2 and 3 log, depending on the Neo-Temper rate applied, with small standard deviations, which shows uniform coverage of Neo- Temper on the wheat kernels during tempering.

Table XIII

Table XIV

Table XV

Example 4

[0150] This example refers to an industrial scale surrogate validation in a wheat flour mill. More particularly, this example refers to the efficacy of treating hard wheat inoculated with Enterococcus faecium NRRL B-2354, a surrogate for Salmonella and Escherichia coli, with 5 L/t Neo-Temper during tempering process.

[0151] The process comprises the following steps:

1) Inoculation:

[0152] According to procedure defined in example 3, approximately 12,000 kg wheat was inoculated using overnight culture of E. faecium NRRL B-2354 at a rate of 6% (60 ml inoculum per kg) followed by drying at 105 °F (40 °C) in fluidized continuous dryer (i.e. fluid bed dryer) at 3 t/h throughput rate. The moisture content of the inoculated wheat was returned to its original value. The inoculum level is shown in table XVIII. The inoculum was applied in the continuous system, like the other inoculations. Overnight inoculum was applied at 60 L/t rate with the Neo-Pure Applicator and then dried in the Neo-Pure dryer at 105 F. The average fresh wheat moisture content was 13.35%, the average fresh wheat water activity was 0.66, the average inoculated wheat moisture content was 14.12% and the average inoculated water activity was 0.75. The inoculated wheat showed having a stable population of E. Faecium for at least two weeks at ambiant temperature (i.e. about 22°C).

[0153] Then the Inoculated wheat was shipped out to a mill facility to be treated during the tempering process and then milled into flour.

2) Pre-treatment quality control :

[0154] A fresh tempering composition was prepared on-site prior to treatment and its strength was confirmed to be within manufacturer’s recommended range using Masters kits that apply Photometric Measuring Technology. More particularly, Neo-Temper composition was prepared by mixing in-line through the static mixer: (i) Neo-Alpha One (i.e based ingredients comprising in water Peracetic Acid (PAA)), and (ii) Neo-Alpha Two (i.e. based ingredient comprising in water Hydrogen Peroxide). Water was further added to obtain the tempering composition. Concentration of the two ingredients used to prepare fresh Neo-Temper was measured before the run using Masters kits that apply Photometric Measuring Technology and were as followed:

Table XVI

3) Treatment:

[0155] The equipment necessary to apply tempering composition on the inoculated wheat was also adjusted and calibrated to ensure accuracy of application rate. Inoculated wheat was sprayed on with Neo-Temper, at 5 L/t using Prominent motor- driven metering pumps. The water application rate fluctuated based on live moisture content reading of wheat and also due to the wheat flow rate as read by the totalizer. Below table shows the Neo-Temper treatment rates, water rates, wheat flow rate, amount of food treated, and tempering duration, for both runs:

Table XVII

4) Tempering:

[0156] Immediately after treatment, wheat was conveyed into tempering silos and stored for 6 h.

5) Milling:

[0157] The treated and tempered wheat was then milled into fine flour at 12.5 - 13.2 t/hr 12.5-13.2 as shown in Table XVII.

6) Sampling:

[0158] Wheat samples were collected before tempering process as inoculated control. Fine flour samples were also taken post milling. The samples were sent to conduct microbiological analysis at Applicant’s laboratories.

7) Micro analysis:

[0159] Enumeration of E. faecium was done at Applicant’s laboratories following the procedure described in the FDA Bacteriological Analytical Manual (BAM) (Andrews and Hammack, 2003). Briefly, each 45-g sample of wheat or flour was mixed with 90 (1:2 w/v) or 180 (1 :4 w/v) ml of buffered peptone water (BPW), respectively, in a sterile stomacher bag, followed by mixing through shaking vigorously 50 times in a 30 cm (1 ft.) arc by hand. Then, the samples were left to stand for 3-5 minutes and shaken vigorously 5 times in a 30 cm arc just before 10-fold serial dilutions in BPW were prepared. Plating was done on Enterococci selective agar (Slanetz & Bartley), followed by incubation at 95 °F (35 °C) for 48 h. Results are reported in log CFU/g. The detection limit was either 2 (0.3 log CFU/g) or 4 CFU/g (0.6 log CFU/g), depending on the dilutions used. ) Microbiological Analysis Results:

[0160] The results are shown in the following tables demonstrate that Neo-Temper was able to reduce the population of E. faecium from 6.69 ± 0.09 to 3.31 ± 0.08 at 5 L/t of Neo-Temper. This indicates that Neo-Temper was able to produce 3.38 log reduction on E. faecium, a surrogate of Salmonella and E. coli. The inoculation technique used for inoculating approx. 12,000 kg of wheat proved to be very effective and efficient with standard deviation of as small as 0.09. Neo-Temper was effective in reducing population of Salmonella and E. coli surrogate by 3.38 log with small standard deviation of 0.08, which shows uniform coverage of Neo-Temper on the wheat kernels during tempering.

Table XVIII Table XIX

Example 5

[0161] This example refers to an industrial scale surrogate validation with wheat flour mill. More particularly, this example refers to the efficacy of treating hard wheat inoculated with Enterococcus faecium NRRL B-2354, a surrogate for Salmonella and Escherichia coli, with 5 and 9 L/t Neo-Temper during tempering process

1) Inoculation:

[0162] Approximately 27,000 kg wheat was inoculated using overnight culture of E. faecium NRRL B-2354 at a rate of 6% (60 ml inoculum per kg) followed by drying at 105 °F (40 °C) in fluidized continuous dryer (i.e. fluid bed dryer) at 3 t/h throughput rate. The moisture content of the inoculated wheat was returned to its original value. The inoculum level was 6.69 Log CFU/g as shown in Table XXII. The inoculum was applied in the continuous system, like the other inoculations. Overnight inoculum was applied at 60 L/t rate with the Neo-Pure Applicator and then dried in the Neo-Pure dryer at 105 F. The average fresh wheat moisture content was 13.35%, the average fresh wheat water activity was 0.66, the average inoculated wheat moisture content was 11.12% and the average inoculated water activity was 0.75. The inoculated wheat showed having a stable population of E. Faecium for at least two weeks at ambiant temperature (i.e. about 22°C).

[0163] Then the Inoculated wheat was shipped out to an industrial mill facility to be treated during the tempering process and then milled into flour.

2) Pre-treatment quality control (QC):

[0164] A fresh tempering composition was prepared on-site prior to treatment and its strength was confirmed to be within manufacturer’s recommended range using Masters kits that apply Photometric Measuring Technology. Neo-Temper composition was prepared by mixing in-line through the static mixer: (i) Neo-Alpha One (i.e based ingredients comprising in water Peracetic Acid (PAA)), and (ii) Neo-Alpha Two (i.e. based ingredient comprising in water Hydrogen Peroxide). Water was further added to obtain the tempering composition. Concentration of the two ingredients used to prepare fresh Neo-Temper was measured before the run using titration kits that apply Photometric Measuring Technology and were as followed: Table XX

3) Treatment:

[0165] The equipment necessary to apply tempering composition on the inoculated wheat was also adjusted and calibrated to ensure accuracy of application rate. Inoculated wheat samples were sprayed on with Neo-Temper. Approximately half of the wheat (13,500 kg) was treated at 5 L/t and the remaining half was treated at 9L/t using Prominent motor-driven metering pumps. Prior to treatment the pumps for both ingredients were calibrated. Below table shows the Neo-Alpha One and Neo-Alpha Two’s flow rates, water flow rates, wheat flow rate, amount of food treated, and tempering duration, for both runs:

Table XXI

4) Tempering:

[0166] Immediately after treatment, the wheat was conveyed into tempering silos and stored for 18 h. 5) Milling:

[0167] The treated and tempered wheat was then milled in an industrial mill into a fine flour at a t/hr throughput as illustrated in the following table XXI.

6) Sampling:

[0168] Wheat samples were collected before tempering process as inoculated control. Fine flour samples were also taken post milling for each run. The samples were sent to Applicant’s laboratories for microbiological analysis. Each sample represents about 1 kg collected into a sterile bag at equally distributed time points.

7) Micro analysis:

[0169] Enumeration of E. faecium was done at Agri-Neo lab following the procedure described in the FDA Bacteriological Analytical Manual (BAM) (Andrews and Hammack, 2003). Briefly, each 45-g sample of wheat or flour was mixed with 90 (1 :2 w/v) or 180 (1 :4 w/v) ml of buffered peptone water (BPW), respectively, in a sterile stomacher bag, followed by mixing through shaking vigorously 50 times in a 30 cm (1 ft.) arc by hand. Then, the samples were left to stand for 3-5 minutes and shaken vigorously 5 times in a 30 cm arc just before 10-fold serial dilutions in BPW were prepared. Plating was done on Enterococci selective agar (Slanetz & Bartley), followed by incubation at 95 °F (35 °C) for 48 h. Results are reported in log CFU/g. The detection limit was either 2 (0.3 log CFU/g) or 4 CFU/g (0.6 log CFU/g), depending on the dilutions used.

8) Microbiological Analysis Results:

[0170] The results are shown in the following tables demonstrating that Neo-Temper was able to reduce the population of E. faecium from 6.47 ± 0.10 to 3.84 ± 0.39 and 3.14 ± 0.09 LogCFU/g at 5 and 9 L/t of Neo-Temper, respectively. This indicates that Neo-Temper was able to produce 2.63 and 3.33 log reduction on E. faecium, a surrogate of Salmonella and E. coli, respectively. The inoculation technique used for inoculating approx. 27,000 kg of wheat proved to be very effective and efficient with standard deviation of as small as 0.10. Neo-Temper was effective in reducing population of Salmonella and E. coli surrogate by 2.63 and 3.33 log, depending on the Neo-Temper rate applied, with small standard deviations, which shows uniform coverage of Neo-Temper on the wheat kernels during tempering. Table XXII

*TFTC: Too Few to Count **TNTC: Too Numerous to Count

Table XXIII

^* TFTC: Too Few to Count **TNTC: Too Numerous to Count Table XXIV

Example 6

[0171] Comparison of two sets of inoculated wheat at different storage temperature. Two sets of five samples of inoculated wheat were collected after step 1) of the above- mentioned Example 4. One set of 5 samples was submitted to storage conditions at ambiant temperature (i.e. about 22°C), and the other set of 5 samples was submitted to storage conditions at fridge temperature (i.e. about 4°C).

[0172] Then micro analysis were carried according to the following procedure: Enumeration of E. faecium was done at Agri-Neo lab following the procedure described in the FDA Bacteriological Analytical Manual (BAM) (Andrews and Hammack, 2003). Briefly, each 45-g sample of wheat was mixed with 90 (1 :2 w/v) ml of buffered peptone water (BPW), respectively, in a sterile stomacher bag, followed by mixing through shaking vigorously 50 times in a 30 cm (1 ft.) arc by hand. Then, the samples were left to stand for 3-5 minutes and shaken vigorously 5 times in a 30 cm arc just before 10-fold serial dilutions in BPW were prepared. Plating was done on Enterococci selective agar (Slanetz & Bartley), followed by incubation at 95 °F (35 °C) for 48 h. Results are reported in log CFU/g. The detection limit was 2 (0.3 log CFU/g) CFU/g.

[0173] Data obtained between September 18 and November 27, were the following:

[0174] Data of September 18 (first set of 5 samples) - are presented in the following table XXV:

Table XXV [0175] Data of September 24 are presented in the following table XXVI and XXVII:

Table XXVI

Table XXVII

[0176] Data of October 1 are presented in the following table XXVIII and XXIX: Table XXVIII

Table XXIX

[0177] Data of October 8 are presented in the following table XXX and XXXI:

Table XXX

Table XXXI

[0178] Data of October 15 are presented in the following table XXXII and XXXIII:

Table XXXII

Table XXXIII

[0179] Data of October 22 are presented in the following table XXXIV and XXXV:

Table XXXIV

Table XXXV

[0180] Data of October 31 are presented in the following table XXXVI and XXXVII: Table XXXVI

Table XXXVII

[0181] Data of November 8 are presented in the following table XXXVIII and XXXIX: Table XXXVIII

Table XXXIX

[0182] Data of November 19 are presented in the following table XL and XLI: Table XL

Table XLI

[0183] Data of November 27 are presented in the following table XLI I and XLI II: Table XLII

Table XLIII

[0184] Comparisons between average results of the first set of samples (ambient temperature) and second sets of samples (fridge temperature) appear in the following tables: Table XLIV

Table XLV

Table XLVI

[0185] Below table XLVI 11 shows the inoculum level on the wheat on week 0 compared to week 7, when stored in ambient conditions. Tables XLIX and L compares data in Table XLVI 11 with ANOVA method and, since the P value is less than 0.05 (it is 0.00306), there is a significant difference between the 2 columns compared, meaning that the inoculum level dropped meaningfully after 7 weeks.

Table XLVI 11 Table XLIX

Table L

[0186] Below table LI shows the inoculum level on the wheat on week 0 compared to week 7, when stored in cold storage conditions. Tables LII and LIIII compare data in Table LI with ANOVA method and, since the P value is less than 0.05 (it is 0.00306), there is a significant difference between the 2 columns compared, meaning that the inoculum level dropped meaningfully after 7 weeks. Table LI

Table Lll

Table LIII

[0187] 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.