CN1713824A - Method for reducing acrylamide in foods, foods having reduced levels of acrylamide - Google Patents

Abstract

A method for the reduction of acrylamide in food products, food products having reduced levels of acrylamide, and an article of commerce. In one aspect, the method comprises reducing the level of asparagine in a food material before heating. In one embodiment, the method comprises extracting at least a portion of the asparagine from the food material before heating. In yet another aspect, an article of commerce communicates to the consumer that a food product has reduced or low of acrylamide or asparagine.

Description

Ways to reduce acrylamide in food, foods containing reduced amounts of acrylamide

field of invention

The present invention relates to reducing the content of acrylamide in foods, and to foods containing reduced amounts of acrylamide. The invention further relates to a commodity.

Background of the invention

Foods containing carbohydrates have been a major part of people's diets since the dawn of civilization. Carbohydrate-containing foods such as breads, breakfast cereals, snacks, biscuits, cookies, French fries, cooked starchy vegetables, taco crusts, and snacks are commonly consumed today. While these foods have been part of people's diets for countless years, researchers have only recently discovered that many of these foods contain acrylamide.

In April 2002, researchers from the Swedish National Food Administration and Stockholm University announced their discovery that acrylamide, a potentially carcinogenic compound, is produced in many types of cooked food. Acrylamide has carcinogenic potential in mice similar to other carcinogens in food, but its relative carcinogenic potential in food is unknown in humans. For acrylamide, only limited population data are available, and these data do not demonstrate a carcinogenic risk from occupational exposure to acrylamide. (FAO/WHO Identification of Acrylamide in Food as a Health Concern: Summary Report; Geneva, Switzerland, 25-27 June 2002)

While further research is needed to determine the health effects, if any, of people consuming acrylamide at levels typically found in these foods, many consumers have expressed concern. It is therefore an object of the present invention to provide a method for reducing the content of acrylamide in food products. The present invention also aims to provide food products containing reduced amounts of acrylamide. Further, the present invention seeks to provide a commercial product which informs consumers that food products contain reduced or low amounts of acrylamide.

Summary of the invention

In one aspect, the present invention provides a method of reducing acrylamide levels in foods. In one embodiment, the method includes reducing the asparagine content of the food material prior to heating.

In another aspect, the present invention provides a method of reducing the level of asparagine in a food material. In one embodiment, the method comprises extracting at least a portion of the asparagine from the food material prior to heating.

In another aspect, the present invention provides food products with reduced amounts of acrylamide.

In another aspect, the present invention provides a food material with reduced asparagine.

In another aspect, the present invention also provides a commercial product which informs the consumer that the food product contains reduced or small amounts of acrylamide or asparagine.

All cited documents are, in relevant part, incorporated herein by reference; the citation of any document is not to be construed as an admission that it is prior art to the present invention.

Brief description of the drawings

Figure 1. Figure 1 outlines the proposed reaction mechanism according to which acrylamide is formed from asparagine and a carbonyl source such as glucose. R ₁ and R ₂ can be H, CH ₃ , CH ₂ OH, CH ₂ (CH ₂ ) _n CH ₃ , or any other components of reducing sugar; n can be any integer less than 10.

Figure 2. Figure 2 outlines the proposed reaction mechanism according to which asparaginase reacts with asparagine to prevent the formation of acrylamide.

Figure 3. Figure 3 lists sample chromatograms for the LC analysis of asparagine and aspartic acid. The x-axis represents retention time and the y-axis represents response.

Detailed description of the invention

Applicants have discovered that asparagine, a naturally occurring amino acid present in nearly all biological systems, can produce acrylamide when heated. Foods containing more asparagine thus tend to contain higher levels of acrylamide when heated; this is especially true when asparagine-containing foods are heated in the presence of reducing sugars. It has also been found that more acrylamide is formed when the food is cooked to a lower final moisture content.

Without being bound by theory, it is believed that acrylamide can be produced in food products by the reaction mechanism outlined in Figure 1 . It is believed that the [alpha]-amino group of free asparagine reacts with a carbonyl source to form a Schiff base. Upon heating, the Schiff base adduct decarboxylates to form a product that:

(1) hydrolysis to form β-alanine amide (this compound can be further degraded to form acrylamide under heating conditions), or (2) decomposition to form acrylamide and the corresponding imine. (Applicants have discovered that the atoms of the cyclic precursor comprise the carbon and nitrogen atoms in acrylamide.)

Accordingly, applicants have further discovered that removal of at least a portion of asparagine prior to cooking reduces the formation of acrylamide in heated foods. When the aforementioned foods containing reduced amounts of asparagine were heated, the amount of acrylamide formed was also reduced. Extraction with a solvent is the preferred method for removing asparagine. The preferred solvent is water.

A. Methods to reduce acrylamide in food

In one aspect, the invention provides a method of reducing acrylamide in a food comprising reducing the asparagine content of the food material prior to final heating (eg, cooking). In one embodiment, the method comprises extracting at least a portion of the asparagine from the food material prior to final heating. In a preferred embodiment, the method for reducing the level of acrylamide in foods comprises:

(1) providing a food material, wherein the food material comprises asparagine;

(2) Optionally reduce the particle size of the food material;

(3) Optionally increase the cell membrane permeability of food materials;

(4) removing at least some asparagine from the food material; and

(5) Heating the food material to form the final food.

In another aspect, the present invention provides a method of reducing asparagine in a food material. In one embodiment, the method comprises extracting at least a portion of asparagine from the food material. In one embodiment, extracting includes blanching the food material in a solvent. The preferred solvent is water. In a preferred embodiment, the method of reducing asparagine content in foods comprises:

(1) providing a food material, wherein the food material comprises asparagine;

(2) Optionally reduce the particle size of the food material;

(3) optionally increases the cell membrane permeability of the food material; and

(4) Extract asparagine from food materials.

1. Providing a food material, wherein the food material comprises asparagine

The term "food material" as used herein includes any type of asparagine-containing edible substance used in the preparation of food, including mixtures of two or more foods.

2. Optionally reduce the particle size of food materials

Optionally, but preferably, the particle size of the food material can be reduced, especially when asparagine removal includes extraction. The extraction process is believed to be a diffusion-controlled phenomenon, so shortening the diffusion distance increases extraction efficiency. Reducing the particle size of the food material reduces the diffusion distance, thereby increasing extraction efficiency. Particle size reduction can be accomplished by any suitable method, including cutting, chopping, macerating, comminuting, grinding, shredding, extruding, pounding, or combinations of these methods.

3. Optionally increase the cell membrane permeability of food materials

Keeping vital components inside living cells is important for cell viability. Many cells use active transport to maintain the concentration of important intracellular components at higher levels than osmosis would allow. Due to this principle, extraction of certain components from cells is difficult. Without being bound by theory, it is believed that asparagine is within the cellular structure of the food material; this makes asparagine less suitable for extraction. Applicants have found that increasing the permeability by altering the structure of the cell membrane can greatly increase the extraction efficiency of asparagine.

The cell membranes of the food material can be altered to enhance asparagine extraction by any suitable method, including, but not limited to, heating (e.g., convection, radiation, microwave, infrared), osmotic pressure changes, changing the pH of the cellular environment , treatment with one or more enzymes (e.g., cellulolytic enzymes such as cellulase, hemicellulase, pectinase, or mixtures thereof), freeze-thaw cycles, other cell membrane disruption methods (e.g., sonication) , or a combination of them.

In a preferred embodiment, blanching is used to alter cell membranes. During blanching, the permeability of cells can be affected in various ways. For example, the contents of the cell may expand (eg, due to starch gelatinization), causing the cell membrane to rupture. In addition, heating can denature cell membrane proteins, causing cells to leak. This leads to an increased extraction efficiency of asparagine.

4. Removal of at least part of asparagine from food material

At least part of the asparagine is removed from the food material. Preferably, the level of asparagine in the food material is reduced by at least about 10%, preferably at least about 30%, more preferably at least about 50%, still more preferably at least about 70%, even more preferably at least about 90%.

Any suitable method for removing asparagine can be used. A preferred method of removing asparagine involves extraction. Extraction as used herein includes any method of contacting a food material with a solvent to remove some of the asparagine. Any asparagine-soluble solvent can be used for extraction (eg, any asparagine-soluble food grade solvent, acid or base), however the preferred solvent is water. Water is an ideal solvent for asparagine extraction for several reasons: (1) asparagine is very soluble in water; (2) water is very inexpensive; and (3) water is generally considered safe.

Any suitable extraction method can be used. For example, extraction may include methods such as soaking, leaching, washing, rinsing, main bath, etc., or a combination of these methods.

Preferably, the extraction process is carried out at a temperature at which the solvent is a liquid and the physical properties of the food material (eg, those that affect the final food product) are not adversely affected. When water is used as the solvent, the lower temperature limit is typically around 0°C; however, if the freezing point is lowered using a salt or co-solvent (or some other means of obtaining freezing point depression), the lower limit can be lower than 0°C. The upper temperature limit is typically below the denaturation temperature of the protein, eg, below about 170°F (77°C). In one embodiment, the extraction temperature is from about 5°C to about 70°C, preferably from about 10°C to about 60°C.

During extraction, not only asparagine is removed, but various other solvent-soluble (eg, water-soluble) components are also removed. Because many of these solvent-soluble components can contain flavor compounds, extraction can negatively affect the flavor of the final food product. Applicants have discovered that the use of the main bath extraction method minimizes the removal of other solvent soluble components, including flavor compounds, from the food material.

Main Bath Extraction Method

The main bath can be used to selectively extract one or more components from a food material without adversely affecting the concentration of other components. A main bath is established by successively extracting batches of food material with a solvent-containing bath to establish or approximate equilibrium of one or more extractable components in the solvent-extracted food material. These components may be selectively separated or removed from the solvent using any suitable method. The remaining soluble components are at or near equilibrium with the food material. Successively, another batch or batches of food material are treated with the defined bath. This results in the selective removal of said components without adversely affecting the concentrations of other combinations. After each batch of food material is processed, additional solvent can be added to maintain a constant bath volume.

Once the main bath is established, successive batches of food material can be extracted in a batch, semi-batch, or continuous manner (eg, a countercurrent continuous process in which food is pumped in one direction and solvent is pumped in the opposite direction).

In the present invention, a main bath can be used to selectively remove asparagine from a food material. The main bath method to remove asparagine from the solvent can be implemented in various ways. For example, these means may include adding the asparagine-converting enzyme to the solvent in the main bath, pumping the solvent through a column immobilized with the asparagine-converting enzyme, or pumping the solvent through a column containing For example, a sorbent column comprising an acceptor site specific for asparagine).

In one embodiment, an asparagine converting enzyme is added to the main bath to selectively remove asparagine. As used herein, "asparagine converting enzyme" or "enzyme" includes any enzyme capable of changing the chemical structure of asparagine. For example, deamidases with asparagine conversion function are included in these terms. As used herein, the terms "asparagine converting enzyme" and "enzyme" include one or more enzymes; for example, the term includes a mixture of two or more enzymes.

In a preferred embodiment, the asparagine converting enzyme is an enzyme capable of hydrolyzing the amide group of free asparagine. A preferred enzyme for use herein is asparaginase. A preferred source of asparaginase is Sigma-Aldrich, catalog #A2925. Without being bound by theory, it is believed that the addition of this enzyme degrades the side chain of asparagine; in doing so, the amide bond is hydrolyzed and the asparagine is converted to aspartic acid. The reaction mechanism is shown in Figure 2.

As the enzymes in the main bath convert asparagine to aspartic acid, this creates a driving force for additional asparagine extraction in subsequent batches of food material. The extractable material is balanced with the food material such that other soluble food material components other than asparagine are not extracted, while asparagine continues to react and be converted by the enzyme. Aspartic acid formed from asparagine leaches back into the food material and equilibrates. Supplementary solvent and/or enzyme-containing solutions are added after each batch of food material to compensate for the solution carried over from the previous batch of food material; this keeps the main bath at constant volume.

Enzymes are sold by activity unit, not by weight or volume. Thus, the effective amount of enzyme required to achieve the desired reduction in acrylamide in the final food product will depend on the activity of the particular enzyme product used.

The amount of enzyme added depends on the amount of asparagine reduction, and thus the desired reduction in acrylamide. The amount of enzyme added also depends on the amount of asparagine contained in the food material; food materials containing more asparagine usually need to increase the amount of enzyme or increase the reaction time to achieve the same reduction of acrylamide. The amount of enzyme added will also depend on the particular enzyme used (eg, the ability of the particular enzyme to degrade asparagine) and the particular food material being treated. Those skilled in the art will be able to determine an effective amount of an enzyme depending on the particular food material, the particular enzyme, the particular activity of the enzyme, and the desired result.

After the enzyme has reacted to the desired extent, it is optionally inactivated or removed from the food material. When using enzymes that are safe for consumption (eg, naturally occurring and found in common foods), it is optional not to inactivate or remove the enzymes. Alternatively, the enzyme may be inactivated using any suitable method of inactivating the enzyme. For example, enzymes can be inactivated by the use of heat, pH adjustment, treatment with proteases, or combinations thereof. Also, enzymes may be removed from the food material using any suitable method, including but not limited to extraction. Enzymes can be inactivated, removed, or a combination of inactivated and removed.

In another embodiment, instead of adding the asparagine converting enzyme directly to the main bath, the extract from the bath is pumped through a bed or column immobilized with the asparagine converting enzyme (described The enzyme can be adsorbed or chemically bound to a substrate (preferably an inert substrate such as a plastic sheet or beads in a column) or to the wall of a hollow membrane tube). The main advantage of this method is that the free enzyme does not come into direct contact with the food material, which would otherwise carry some of the enzyme from the main bath, requiring supplementation. Immobilizing the enzyme reduces or eliminates the expense of supplementing expensive enzymes. This embodiment also has the advantage of minimizing the removal of other solvent soluble components from the food material.

In another embodiment, the solvent stream from a defined main bath is sent to a column containing hollow fiber membranes (such as those disclosed in U.S. Patent No. 5,869,297), dialysis material, or size exclusion material (e.g., zeolites). In , the size exclusion material allows asparagine molecules or other molecules of equal or smaller size to diffuse out of the liquid stream. The net effect is the selective removal of asparagine from the food material to achieve the desired reduction in acrylamide without detrimentally reducing the flavor of the final food product.

In another embodiment, the extract stream is sent to a column comprising an adsorbent that selectively absorbs asparagine. Suitable adsorbents may include, but are not limited to, molecular sieves, zeolites, cyclodextrins, clays, diatomaceous earth, silica (e.g., magnesium silicates such as ^Florisil® ), ion exchange resins (anionic or cationic or mixed resins, such as Amberlite ^(R ), or combinations thereof. The net effect is the selective removal of asparagine from the food material to achieve the desired reduction in acrylamide without detrimentally reducing the flavor of the final food product.

In another embodiment, the extract stream is sent to a column comprising an asparagine specific absorbent comprising receptor sites specific for asparagine. The net effect of this approach is the selective removal of asparagine from the food material to achieve the desired reduction in acrylamide without detrimentally reducing the flavor of the final food product.

Although the various embodiments described above have been described in terms of columns, it should be understood that these embodiments can also be realized in any other suitable manner, such as fluidized bed, industrial-scale continuous liquid chromatography (for example, such as published 4,210,594, issued to Logan et al. on July 1, 1980), or in a batch fashion where the absorbent is added to the liquid stream and then separated from the liquid stream.

Additionally, although the above embodiments describe a batch process for asparagine removal by treating the food material with a main bath, it should be understood that the food material and solvent can be used in a semi-batch or continuous manner (e.g., a counter-current continuous process in which the food material flows in one direction pumped in while the solvent is pumped in the opposite direction) to contact. Alternatively, the food material can be a bed in a column through which the solvent is pumped.

5. Heating the food material to form the final food

The food mass can then be heated in the usual manner, such as baked, fried, extruded, dried (eg, by means of a vacuum oven or tumble dryer), puffed or microwaved. In embodiments where the enzyme contacts the food material, the enzyme may be inactivated during the heating process, so the optional inactivation step and the heating (eg, cooking) step can be performed simultaneously. Heat treatment via cooking can denature and inactivate enzymes so that the food material no longer has sustained enzymatic activity. Also, in the heating step, at least a part of the time is given to allow the enzyme to react.

As used herein, "final food" includes, but is not limited to, edible food and food used as an ingredient in making other food.

Preferably, the level of acrylamide in the final food product is reduced by at least about 10%, preferably at least about 30%, more preferably at least about 50%, still more preferably at least about 70%, and even more preferably at least about 90%.

B. Means of implementing the method

The invention can be implemented in any suitable way. For example, the process of the invention can be carried out in a batch, semi-batch or continuous manner.

C. Foods containing reduced amounts of acrylamide

The methods herein can be applied to the production of any suitable food, including, but not limited to, carbohydrate-containing foods that are prepared with heat, especially low moisture foods (eg, less than about 10% moisture). For example, the method can be used to reduce the presence of carbs in mashed potatoes, potato chips, processed snacks, French fries, breakfast cereals, bread, cookies, biscuits, cakes, pizza crust, pretzels, hash browns, shredded potato balls, corn Acrylamide content in tortilla and taco crusts.

In one embodiment, the fried processed potato chips have an acrylamide content of less than about 400 ppb, preferably less than about 300 ppb, more preferably less than about 200 ppb, still more preferably less than about 50 ppb, even more preferably less than about 10 ppb.

In another embodiment, the crisps have an acrylamide content of less than about 40 ppb, preferably less than about 30 ppb, more preferably less than about 20 ppb, even more preferably less than about 10 ppb, and most preferably less than about 5 ppb.

In another embodiment, French fries made from chipped potatoes have an acrylamide content of less than about 40 ppb, preferably less than about 30 ppb, more preferably less than about 20 ppb, and most preferably less than about 10 ppb.

In another embodiment, the corn snack has an acrylamide content of less than about 75 ppb, preferably less than about 50 ppb, more preferably less than about 10 ppb.

Although the methods herein will generally be described in terms of the preferred dehydrated potato products, processed French fries, potato chips, french fries, and corn snacks, those skilled in the art will appreciate that the methods herein can be applied to any suitable product. Non-limiting examples include biscuits, breads (such as rye, wheat, oats, potatoes, white whole grain products, all purpose flour, barley cakes, twist breads, buns, rolls, pita, matzo , Italian herb bread, toasted croutons, double toasted croutons, croutons, soft muffins, soft and firm breadsticks, heated ready-to-eat breads), baked cakes, cookies, Danish pastries, croissants , flan, pie crust, pie crust, muffins, brownies, baguettes, donuts, snacks (such as pretzels, nachos, corn chips, potato chips, processed snacks, processed chips, Extruded snacks, extruded filled snacks, dry meals on the go, granola, muesli, crisps), flour, cornmeal, mixes (such as cake mix, cookie mix, brownie mix , bread mix, pancake mix, pancake mix, custard batter mix, pizza dough), frozen dough (e.g. biscuits, bread, breadsticks, croissants, meal kits, pizza dough, cookies, Danish Meringues, brownies, pie crusts), frozen foods (e.g. pie crusts, pies, flan, wraps, pizza, food crusts, cakes, French fries, hash browns, breaded foods such as chicken and fish, breaded vegetables), bagels, breakfast cereals, snacks, chips, vegetables (such as dried, baked, baked, fried, fried, vacuum-dried), Taco crust, hash browns, mashed potatoes, toast, toasted sandwiches, flour and tortillas, pancakes, pancakes, waffles, batter, pizza crust, rice, nut-based foods (such as peanut butter, foods containing chopped nuts), fruit (such as dried, roasted, baked, fried, fried, vacuum-dried, baked, jelly, pie filling, burnt, raisins, cranberries, cherries), fried fried fruit, alcoholic beverages (such as beer and ale), foods containing roasted cocoa beans (such as dog food, cat food, ferret food, guinea pig food, gerbil food, hamster food, Bird Feed, Camel Feed, Ostrich Feed, Emu Feed, Cattle Feed, Deer Feed, Elk Feed, Buffalo Feed, Rabbit Feed, Rat Feed, Rat Feed, Chicken Feed, Turkey Feed, Pig Feed, Horse Feed, Goat Feed, sheep feed, monkey feed, fish feed).

1. Dehydrated potato products containing reduced amounts of asparagine and acrylamide

The present invention can be used to produce dehydrated potato products containing reduced amounts of acrylamide by reducing the asparagine content of the food material. A preferred method of making the above-described dehydrated potato products will be described below, but the invention is not limited to this particular embodiment. Although this embodiment, detailed below, describes extraction of asparagine prior to drying of cooked potatoes, it should be understood that extraction of enzymatic asparagine may be accomplished at any suitable step in the process of making a dehydrated potato product. For example, extraction of asparagine may be performed prior to cooking, post-cooking, pre-shredding, post-shredding, or any other suitable processing step prior to forming the final dehydrated potato product. The methods herein may also be practiced in conjunction with any method known in the art suitable for making dehydrated potato products, as described in "Potato Processing", 4th Edition, edited by Talburt and Smith, AVI Books, Van Nostrand Reinhold Co., New York, 1987, [hereinafter "PotatoProcessing"]) on pages 535 to 646.

In a preferred embodiment, dehydrated potato products, such as potato flakes, potato cakes or potato granules, can be produced according to the following method. Typically, this method includes:

(1) Provide potatoes;

(2) Optionally reduce the particle size of potatoes;

(3) Optionally increase the permeability of potato cell membranes;

(4) Cooking potatoes;

(5) Asparagine is extracted from potatoes;

(6) Moist mashed potatoes made from potatoes; and

(7) Drying the wet mashed potatoes to form a dehydrated potato product.

It should be understood that although the extraction step is listed as step (5) in the above embodiments, the extraction step may be performed in any other suitable step of the method. In another embodiment, a method of making a dehydrated potato product comprises:

(1) Provide potatoes;

(2) Optionally reduce the particle size of potatoes;

(3) Optionally increase the permeability of potato cell membranes;

(4) Cooking potatoes;

(5) Make wet mashed potatoes with potatoes;

(6) drying the wet mashed potatoes to form a dehydrated potato product; and

(7) extract asparagine from potato, wherein said extraction is carried out in the above steps 1 to 6

before, during or after any of the

It should be understood that steps 1 to 6 above may be performed in any suitable order.

Any suitable potato, such as those used to make conventional potato chips, cakes or granules, can be used to make the dehydrated potato products herein. Preferably, varieties such as, but not limited to, Norchip, Norgold, Russet Burbank, Lady Rosetta, Norkotah, Sebago, Bintje, Aurora, Saturna, Kinnebec, IdahoRusset, Altura, Russet Norkotah, Atlantic, Shepody, Asterix and Mentor can be used potatoes to make dehydrated potato products.

Potatoes that contain less than about 5% reducing sugars (calculated on a dehydrated potato basis), preferably less than about 3%, more preferably less than about 2% are preferred. For example, potatoes that contain low levels of reducing sugars (ie, less than 1.5%) are especially preferred for making dehydrated potato products for making fried potato snacks.

Potatoes are steamed to soften them for mashing. Potatoes can be peeled, partly peeled or left unpeeled. Potatoes may be whole or cut into slices of any size prior to cooking. The cooking operation can be any heat or other type of cooking method that softens the potatoes to mash them. For example, potatoes can be cooked by submerging them in water or steam.

For example, typically, potato chips with an average thickness of about 0.95 cm (3/8 inch) to about 1.27 cm (1/2 inch) can be used at temperatures from about 200°F (93°C) to about 250°F (121°C) The steam cooking is about 12 minutes to about 45 minutes, more specifically about 14 minutes to about 18 minutes. Typically, shredded potato chips can be steam-cooked at a temperature of about 200°F (93°C) to about 250°F (121°C) for 7 minutes to about 18 minutes, more specifically about 9 minutes to about 12 minutes to achieve desired firmness.

Asparagine in cooked potatoes (eg, cooked potato chips) can be extracted with water. Optionally, the cooked potato pieces can be reduced in size by the various methods described above for extraction processing. Boiled potatoes may be extracted by soaking in water for a predetermined period of time. Extraction times can vary from minutes to hours, depending on the amount of asparagine reduction desired. Typical extraction times are from about 30 minutes to about 4 hours. After the extraction phase, the potatoes are separated from the extraction solution. This can be accomplished by any suitable method such as filtration, centrifugation or decantation.

Following the extraction process, post-extraction comminution of the potatoes may be accomplished by any suitable method such as, but not limited to, milling, mashing, chopping or combinations thereof to form a wet mashed potato.

Optional ingredients can be added and mixed into the wet mashed potatoes. The aforementioned optional ingredients may include starch. The starch may include, but is not limited to, any suitable native or modified starch, including any dried potato product that is added or added back to mashed potatoes. Emulsifiers are also optionally added to the wet mashed potatoes as processing aids.

Once the mashed potatoes have been formed, they can be further dried and processed to form dehydrated potato products as described below. These dehydrated potato products may be in any shape such as, but not limited to, flakes, cakes, granules, agglomerates, flakes, shreds, flakes, flour or granules. Alternatively, wet mashed potatoes can be used to make products such as, but not limited to, mashed potatoes, potato patties, potato scones, and potato snacks (such as pressed French fries, potato sticks, and potato chips). For example, wet mashed potatoes can be used to make French fries, such as those described in US Patent 3,085,020, issued April 9, 1963 to Backinger et al.

Any suitable method of making the above-described dehydrated potato products from mashed potatoes, such as those known in the art, may be employed and any suitable equipment may be used. For example, mashed potatoes may be dried into flakes according to known methods, such as those described in U.S. Patent 6,066,353 issued May 23, 2000 to Villagran et al., and 1956 US Patent 2,759,832 issued August 19, 1957 to Cording et al., and US Patent 2,780,552 issued February 5, 1957 to Willard et al. Mashed potatoes may be dried into cakes as described in US Patent 6,287,622, issued September 11, 2001 to Villagran et al. According to the method described in U.S. Patent 3,917,866 issued November 4, 1975 to Purves et al., or by other known methods (such as in U.S. Patent 2,490,431 issued December 6, 1949 to Greene et al. Those mentioned above), can be made into granules by processing said mashed potatoes. Suitable dryers may be selected from those well known drying apparatus including, but not limited to, fluid bed dryers, scraped wall heat exchangers, tumble dryers, freeze dryers, airlift dryers, and the like.

Preferred drying methods include those that reduce the overall heat input. For example, when making dehydrated potato chips, freeze drying, drum drying, resonance or pulsed flow drying, infrared drying, or combinations thereof are preferred; when making dehydrated potato granules, air lift drying, fluidized bed drying or their combination is preferred.

Although the dehydrated potato products herein are described primarily in terms of potato chips, it will be apparent to those skilled in the art that the mashed potatoes of the present invention can be dehydrated to form any desired dehydrated potato product derived from mashed potatoes.

Drum drying (for example using a drum dryer commonly used in the potato product industry) is the preferred method of drying mashed potatoes to form flakes. A preferred method utilizes a single drum dryer in which wet mashed potatoes are spread onto the drum in flakes, the flakes having a thickness of about 0.005" to about 0.1", preferably about 0.005" to about 0.05", more preferably about 0.01". Generally, when using a drum dryer, mashed potatoes are added to the upper surface of the drum by conveying means. Small diameter unheated rolls gradually apply fresh mashed potatoes to the portion already on the drum, whereby A sheet or layer of predetermined thickness is formed. The peripheral speed of the small roller is the same as that of the drum. After the mashed potato layer rotates along a part of the drum's circumference, the scraper strips the dried flakes from the drum to remove the dried flakes. Typically, the drum dryer itself is heated to about 250°F (121°C) by pressurizing the steam contained within the drum to about 483 kPa (70 psi) to about 965 kPa (140 psi). ) to about 375°F (191°C), preferably about 310°F (154°C) to about 350°F (177°C), more preferably about 320°F (160°C) to about 333°F (167°C) Temperature. For best results, the rotational speed of the dryer drum and its internal temperature are properly controlled so as to obtain a final product with a moisture content of from about 5% to about 14%, preferably from about 5% to about 12%. Typically, about 9 A rotational speed (sec/rev) of from about 11 sec/rev to about 20 sec/rev is sufficient.

Once the wet mashed potatoes are flaked and dried, the resulting dried flakes of potato chips can be broken into smaller pieces if desired. These smaller pieces can be of any desired size. Any method of breaking the flakes that minimizes starch and potato cell damage can be used, such as crushing, grating, mincing, cutting or shredding. For example, the flakes can be crushed with a Urschel Comitro manufactured by Urschel Laboratories, Inc. (Valparaiso, Indiana) to break the flakes. Alternatively, the potato chips can be left whole. As used herein, both whole potato slices and smaller sliced portions are included in the term "potato slices".

2. Foods made from dehydrated potato products containing reduced amounts of acrylamide and asparagine Taste

Dehydrated potato products containing reduced amounts of acrylamide and asparagine may be used to make any suitable food product. A particularly preferred use of dehydrated potato products is in the manufacture of processed potato chips made from dough. Examples of the aforementioned processed potato chips include: U.S. Patent 3,998,975 issued December 21, 1976 to Liepa; U.S. Patent 5,464,642 issued November 7, 1995 to Villagran et al; Those in US Patent 5,464,643 to Lodge and PCT Application PCT/US95/07610 published by Dawes et al. on January 25, 1996 as WO 96/01572.

Dehydrated potato products can also be rehydrated and used to make food products such as mashed potatoes, potato pies, potato pancakes and other potato snacks such as French fries and chips. For example, dehydrated potato products such as those described in U.S. Patent 3,085,020 issued April 9, 1963 to Backinger et al. and U.S. Patent 3,987,210 issued October 18, 1976 to Cremer can be used to make press fried potato products . Dehydrated potato products can also be used in bread, thickened broths, sauces, baby food or any other suitable food.

3. Potato chips with reduced acrylamide

The present invention can be used to make potato chips containing reduced amounts of acrylamide. A preferred method of making the potato chip product described above is set forth below, but the invention is not limited to this particular embodiment. A typical method for making potato chips is described in "Potato Processing", pages 371 to 489.

In a preferred embodiment, the present invention provides a method of reducing acrylamide content in potato chips, the method comprising:

(1) Potatoes are optionally peeled;

(2) Potatoes are optionally washed;

(3) Potato slices are made into potato flakes;

(4) Potato slices are optionally rinsed;

(5) Potato slices are optionally blanched;

(6) extract potato flakes to reduce asparagine content;

(7) Potato flakes are optionally dried;

(8) Fried potato flakes to make potato chips.

Most preferably, the potato flakes are blanched prior to asparagine extraction. Although the extraction of asparagine in step (6) above is described above, it should be understood that the extraction can be performed at any suitable step of the method. In another embodiment, a method of reducing acrylamide levels in potato chips comprises:

(1) Potatoes are optionally peeled;

(2) Potatoes are optionally washed;

(3) Potato slices are made into potato flakes;

(4) Potato slices are optionally rinsed;

(5) Potato slices are optionally blanched;

(6) Optionally dried potato flakes;

(7) Fried potato flakes to make potato chips.

(8) Extraction of potato flakes to reduce asparagine content, wherein said extraction is done before, during or after any one of steps 1 to 7 above.

It should be understood that steps 1 to 7 above may be performed in any suitable order.

The extraction step can be accomplished by any suitable method. Preferred methods may include soaking, extraction with a main bath and rinsing.

In one embodiment, potato flakes having a thickness of about 0.5 mm to about 1.5 mm are used to make potato chips. Scald the slices in water to about 54°C (130°F) to about 77°C (170°F) for about 15 seconds to about 3 minutes. The blanched flakes are optionally cooled. The blanched slices are then soaked in water for about 15 minutes to about 4 hours to extract it. Extraction can be done in one or more extraction steps. The resulting potato flakes have a reduced asparagine content. The potato flakes are then optionally dried before frying to form potato chips.

Potato chips made according to the method of the present invention have an acrylamide content of less than about 40 ppb, preferably less than about 30 ppb, more preferably less than about 20 ppb, even more preferably less than about 10 ppb, and most preferably less than about 5 ppb.

4. French fries

The present invention can be used to make French fries containing reduced amounts of acrylamide. A preferred method of making the above-described French fries will be described below, but the invention is not limited to this particular embodiment. For example, asparagine extraction can be performed at any suitable processing step in a method of making French fries known in the art, such as those described in "Potato Processing" on pages 491 to 534, or described in U.S. Patent Nos. 6,001,411 and 6,013,296 methods.

In a preferred embodiment, the present invention provides a method for reducing the content of acrylamide in French fries, the method comprising:

(1) Potatoes are optionally peeled;

(2) Potatoes are optionally washed;

(3) peeling and cutting potatoes to make potato strips;

(4) Potato fries are optionally rinsed;

(5) Optionally blanched or optionally par-fried potato chips;

(6) Optional cooling of potato chips;

(7) Asparagine is extracted from potato strips;

(8) Potato chips are optionally dried;

(9) Optionally coat potato chips; and

(10) Half-fried potato fries, made into half-finished French fries.

Most preferably, the potato strips are blanched prior to asparagine extraction. Although the extraction of asparagine in step (7) above is described above, it should be understood that the extraction can be performed at any suitable step of the method. In another embodiment, the method of reducing the level of acrylamide in french fries comprises:

(1) Potatoes are optionally peeled;

(2) Potatoes are optionally washed;

(3) peeling and cutting potatoes to make potato strips;

(4) Potato fries are optionally rinsed;

(5) Optionally blanched or optionally par-fried potato chips;

(6) Optional cooling of potato chips;

(7) Potato chips are optionally dried;

(8) Optionally coat potato chips;

(9) Half-fries, made into half-fries; and

(10) Extracting asparagine from potato strips, wherein said extraction is performed before, during or after any one of steps 1 to 9 above.

It should be understood that steps 1 to 9 above may be performed in any suitable order.

The extraction step can be accomplished by any suitable method. Preferred methods may include soaking, extraction with a main bath and rinsing.

The semi-finished french fries are then frozen, packaged and stored for later frying to make finished french fries. The term "potato fries" as used herein is broad enough to include potatoes of any suitable shape, such as potato wedges, waffle fries, curly fries, potato nuggets, hash browns, chunky fries, potato skins, or any other potato part.

Most preferably, the potato strips are blanched before the asparagine is extracted. If it is desired to coat the french fries, a suitable coating material such as starch or a mixture of materials including one or more starches may be used to coat the fries prior to par-frying.

French fries made from the semi-finished French fries of the present invention have an acrylamide content of less than about 40 ppb, preferably less than about 30 ppb, more preferably less than about 20 ppb, most preferably less than about 10 ppb.

5. Corn Snacks

In another embodiment, the corn snack may have an acrylamide content of less than about 75 ppb, preferably less than about 50 ppb, more preferably less than about 10 ppb. Preferred corn snacks include nachos and nachos. Although the methods herein are generally described in terms of the preferred nachos, it should be understood that the methods can be used to make any suitable corn snack.

Nachos are a particularly popular consumer snack product. Nachos are traditionally made from whole corn that has been steamed in hot lime water for about 5 to about 50 minutes and then soaked overnight. This cooking-soaking method softens the husk and partially gelatinizes the starch in the corn endosperm. This cooked-soaked corn (called "nixtamal") is then washed to remove the husk and ground to form a plastic dough (called "dough"), which contains about 50% moisture. The freshly ground dough is sheeted, cut into dim sum pieces, and baked at about 302°C (575°F) to 600°F (302°C to 316°C) for about 15 to about 30 seconds to The moisture content is reduced to about 20% to about 35%. The baked snack dough is then fried in hot oil to form nachos with a moisture content of less than about 3%. See, e.g., U.S. Patent 2,905,559 issued November 1, 1958 to Anderson et al. and U.S. Patent 3,690,895 issued September 12, 1972 to Amadon et al., and "Corn: Chemistry and Technology" (American Association of Cereal Chemists , Stanley A. Watson et al. eds.) pp. 410-420 (1987).

Nachos can also be made from dry dough flour. In typical processes for making the above-mentioned dry dough flours, such as described in U.S. Patent No. 2,704,257 issued March 1, 1955 to de Sollano et al. and U.S. Patent No. 3,369,908 issued February 20, 1968 to Gonzales et al. Of those, limed corn is ground and dehydrated to form a steady state. The dry dough flour can then be rehydrated with water to form a dough dough which can then be used to make nachos, such as those described in WO 01/91581 published December 6, 2001 by Zimmerman et al.

In one embodiment, a corn snack can be prepared by a method comprising:

(1) providing a dough, wherein the dough comprises a dough with reduced asparagine;

(2) dim sum bases made from dough; and

(3) Cook the dim sum base to make corn snacks.

In another embodiment, a corn snack may be prepared by a method comprising:

(1) Optionally blanching the corn;

(2) extracting corn to form asparagine-reduced corn;

(3) Nixtamal is made from corn;

(4) make dim sum base with nixtamal; and

(5) Cook the dim sum base to make corn snacks.

Corn snacks that can be made with the methods herein include nachos, nachos, or extruded corn snacks. Suitable cooking methods may include baking, frying, extruding, and combinations thereof.

It should be understood that extraction can be performed at any suitable step in the process. In one embodiment, a method of making a corn snack comprises:

(1) Optionally blanching the corn;

(2) Nixtamal is made from corn;

(3) make dim sum base with nixtamal;

(4) cooking snack bases to make corn snacks; and

(5) Extracting corn to obtain asparagine-reduced corn, wherein the extraction is performed before, during or after any one of steps 1 to 4 above.

D. Commodities

Another embodiment of the invention is a commodity comprising:

(a) food products, wherein the food products contain reduced amounts of acrylamide;

(b) containers for food; and

(c) Prompt information related to the container.

The notice informs consumers that the food contains reduced amounts of acrylamide. Appropriate reminders include, but are not limited to, reminders for "reduced" or "small" acrylamide, reminders for acrylamide containing less than a specified amount (such as less than 5 ppb), and reminders for foods that meet or exceed the recommended amount or Prompt information for standard quantities (such as specified thresholds or exceeding normal content). In one embodiment, the cue message informs the consumer that the food product is prepared with ingredients that contain reduced or low amounts of asparagine, thereby implying that the food product therefore contains reduced or low amounts of acrylamide.

In another embodiment, the commodity includes:

(a) foods, wherein the foods contain reduced amounts of asparagine;

(b) containers for food; and

(c) Prompt information related to the container.

The notice informs the consumer that the food contains reduced or small amounts of asparagine.

The reminder may be printed matter attached directly or indirectly to the container, attached directly or indirectly to the vicinity of the container, or alternatively may be a printed, electronic or broadcast reminder associated with the container.

Any container in which food can be served, presented, displayed or stored is suitable. Suitable containers include, but are not limited to, bags, cans, boxes, bowls, trays, tubs and cartridges.

Analytical method

Specific analytical methods are used to quantify the parameters characterizing the elements of the invention. These methods are described in detail below.

1. Acrylamide

Method for determination of acrylamide (AA) in food

overview

1- ¹³ C-acrylamide ( ¹³ C-AA) is incorporated into food and then extracted with hot water. The aqueous supernatant was extracted three times with ethyl acetate, then the ethyl acetate extracts were combined and concentrated before analysis by LC/MS with selective ion monitoring specifically detecting AA ^and13C -AA.

extract sample

1. Weigh 6.00±0.01g sample and put it in a 125ml Erlenmeyer flask. NOTE: Place the sample in a food processor and pulse for 30 seconds to achieve a particle size of approximately 0.3 cm (1/8 inch) or smaller. If the sample is too small to be comminuted effectively in a food processor, place the sample in a new plastic bag (such as a Whirl-Pak ^TM or equivalent) and pulverize with a rubber hammer until the particle size reaches approximately 0.3 cm (1/8 inch) or less.

2. Using an adjustable 1000 μL pipette (calibrated), add 120 μL of 100 ng/ ^μL13C -AA in deionized distilled water (ISTD2) directly onto the sample.

3. Using a dispenser, add 40 mL of deionized distilled water to the flask and cover with foil.

4. Place in a water bath at 65°C for 30 minutes.

5. Add 10 mL of 2-dichloroethane to the flask with a dispenser, then homogenize with Tekmar Tissumizer ^™ (SDT-1810) or Ultra- ^Turrax® (T18 Basic) for 30 seconds, or until homogeneous. Rinse the probe with deionized distilled water, and the wash solution is transferred to the flask.

6. Place 25 g of the homogeneous mixture into an 8 dram vial.

7. Cap the tube tightly, then centrifuge at a speed of 2500 to 5200 rpm for 30 minutes.

8. Carefully transfer 8 g of supernatant to another 8 dram vial, being careful not to transfer solid particles there.

9. Add 10 mL of ethyl acetate with a dispenser, cap, and vortex for 10 seconds.

10. Break all emulsions; this can be done by vortexing or shaking once or twice, then separate the layers.

11. Transfer as much of the upper layer (ethyl acetate) as possible to the scintillation vial without transferring any liquid at the interface (water). Extract more than two times with 5 mL of ethyl acetate each time, and add to the same scintillation vial. Then, about 2 g of anhydrous sodium sulfate was added.

12. Concentrate the extract to about 1 ml with a gentle stream of nitrogen in a water bath at 60°C to 65°C. Transfer the extract to a Pierce REACTI-VIAL ^™ or equivalent Erlenmeyer glass flask and further concentrate the extract to a final volume of approximately 100 μL to 200 μL. This extract was placed in an autosampler vial with a conical cannula.

Preparation of standards

Stock solutions and internal standards

the solution weight volumetric flask solvent Concentration (ppm) stock solution 1 0.1000g acrylamide (AA) 100mL ethyl acetate 1000 ISTD 1 0.0100g ¹³ C-acrylamide 100mL ethyl acetate 100 stock solution 2 0.1000g acrylamide (AA) 100mL deionized distilled water 1000 ISTD 2 0.0100g ¹³ C-acrylamide 100mL deionized distilled water 100

intermediate standard the solution Volume of stock solution 1AA (μL) Volumetric flask (mL) solvent Concentration (ppm) INT1 100 10 ethyl acetate 10 INT2 1000 10 ethyl acetate 100

calibration standard standard Volume INT1(μL) Volume INT2(μL) Volume of ISTD1 (μL) Volumetric flask (mL) Solvent Concentration AA(ppm) Concentration ISTD1(ppm) 0 0 0 450 10 ethyl acetate 0 4.50 0.25 250 0 450 10 ethyl acetate 0.250 4.50 0.75 750 0 450 10 ethyl acetate 0.750 4.50 1.5 0 150 450 10 ethyl acetate 1.50 4.50 3.0 0 300 450 10 ethyl acetate 3.00 4.50 5.0 0 500 450 10 ethyl acetate 5.00 4.50

Homogenizer cleaning method

Clean the homogenizer with this cleaning method between the determinations of each sample.

1. Pour hot tap water into a 1 L Erlenmeyer flask (≈80% full), then add one drop of Dawn ^™ Dishwashing Liquid (available from Procter & Gamble Co.) or equivalent.

2. Insert the probe of the dispersing element into the water as far as possible.

3. Homogenize the solution for about 10 to 15 seconds.

4. Pour the wash solution out of the Erlenmeyer flask; rinse with hot tap water and refill the Erlenmeyer flask.

5. Homogenize again for about 10 to 15 seconds.

6. Empty the Erlenmeyer flask and fill it with hot tap water; homogenize again for about 10 to 15 seconds.

7. If the water is not clear and has particulate matter, continue to homogenize clean hot tap water as many times as necessary until this condition is reached.

8. When the hot tap water is clear and free of particulate matter, rinse the probe with deionized distilled water. Analysis by LC/MS

Samples were analyzed using a Waters 2690 LC connected to a Micromass LCZ mass spectrometer. mobile phase 100% _H2O , 10mM _NH4Ac , pH adjusted to 4.6w/ formic acid Chromatographic column 2.0mm×150mm, YMC C18 AQ (purchased from Waters Corp.) flow rate 0.2ml/min interface direct (no layering) Injection volume 5μL MS ionization method Electrospray, positive ion mode MS detection method Selected ion monitoring: m/z 72 (AA), m/z 73 ( ¹³ C-AA); residence time: 0.5s

data analysis

For five standard samples in a series of ethyl acetate, the response ratio (area of AA peak/area of ¹³ C-AA peak) was plotted against the corresponding concentration ratio. All standard samples contained 4.5 μg/mL of ¹³ C-AA, and the concentration of AA was from 0 μg/mL to 5 μg/mL. Linear regression generates a standard curve by means of which the concentration ratios in the extracts are determined from the determined response ratios. When this concentration ratio is multiplied by the precisely ^known13C -AA content (nominally 2 ppm) added to the sample in step 2 of the extraction method, the ppm content of AA is obtained.

Sample calculation for LC/MS:

A standard curve was generated by plotting the response ratio (area of m/z 72/area of m/z 73) on the y-axis against the concentration ratio ([AA]/[13C-AA]) on the x-axis. For this example, the equation for this line is y=0.899x+0.0123.

Measured area of AA peak (m/z 72) at 4.0 minutes: 100,000

Measured area of 13C-AA peak (m/z 73) at 4.0 minutes: 500,000

Response ratio _Rr = 0.200. From the slope and intercept of the standard curve, calculate the concentration ratio R _c :

R _c =(0.200-0.0123)/0.899=0.209

Known the incorporation amount (2ppm) of 13C-AA in the sample, then the measured content of AA is

0.209×2ppm＝0.418ppm

Quality Assurance/Quality Control (QA/QC)

1. All balances used in the preparation of standards and/or samples must have their calibration checked weekly with a series of standard weights. These balances should be checked with at least three weights covering the range of sample/standard weights to be measured.

2. A six-point calibration curve should be made every day.

3. A working reference material (WRM) should be analyzed using each set of samples. The running mean of the concentration of the substance should be within 2σ. If not, the instrument should be recalibrated and the WRM recalculated.

2. Asparagine

Determination of Asparagine and Aspartic Acid in Food and Beverage Products

principle

The weighed sample was mixed with 5% HCl and heated for 30 minutes, then homogenized. A portion of the homogeneous mixture was centrifuged, and a portion of the supernatant was diluted and treated with FMOC reagent (9-fluorenylmethyl chloroformate), which reacts with asparagine and aspartic acid to form highly fluorescent derivatives. FMOC-Asparagine was then separated from other sample matrix components by reverse phase HPLC. Excitation at 260 nm, detection of fluorescence emission at 313 nanometers (nm). Analysis of standards of known concentration allows quantification.

Linearity

A working standard curve of four standard samples (50-600 ppm) gave a correlation of 0.998 or better. The curve derived from the 2000 ppm sample also gave a correlation of 0.998.

precision

Potato products:

Potato starch was spiked with four levels of asparagine and aspartic acid (40, 200, 400 and 600 ppm). The recovery of asparagine was 100% (relative standard deviation less than 4%) and the recovery of aspartic acid was 110% (relative standard deviation less than 4%).

references

1. Herbert, P.; Santos, L; Alves, A. Journal of FoodScience (2001), 66(9), 1319-1325.

2. Heems, Dany; Luck, Geneviewe; Fraudeau, Chrisophe; Verette, Eric. Journal of Chromatography, A (1998), 798(1+2), 9-17.

System repeatability

The working reference material of potato chips was determined in duplicate in 5 days. The result is as follows:

ug/g asparagineug /g aspartic acid

Average 7832.07 1440.98

STD 625.59 195.80

%RSTD 7.99 13.59

The following chemicals and instruments are recommended; however, substitutions of equivalent substances are permitted.

chemical

Water, HPLC grade or Milli-Q ^TM grade

(Millipore)

Acetonitrile, HPLC grade Burdick & Jackson

#AH015-4

Methanol, HPLC grade Fisher #A452-4

Ethyl acetate Baker #9280-3

Pentane Burdick & Jackson

#GC312-4

Asparagine Monohydrate EM Science

Aspartic Acid Sigma #A-8949

Aminoisobutyric acid Sigma #A-8379

9-Fluorenyl chloroformate (FMOC) ICN #150200

Sodium borate EM Science #SX 0355-

1

Boric acid Fisher#A-73

Sodium Bicarbonate ICN#194847

Tetramethylammonium Chloride Fisher #04640-500

Sodium citrate MCB #SX445

Anhydrous citric acid Baker #0122-01

Acetone Burdick & Jackson

#010-4

Hydrochloric acid, 0.1N Fisher #SA48-500

Calcium Chloride Dihydrate Aldrich #22, 350-6

equipment

Pipette, polyethylene (Samco #222)

Volumetric flask (25, 100, 250, 1000ml)

Constant volume pipette (10ml)

Measuring cylinder (100-1000ml)

HPLC reservoir (500ml, 1L or 2L)

beaker

Magnetic Stirrer/Stir Bar

Analytical balance (4 digits)

scintillation tube

Centrifuge tube, screw cap with lid (100×16mm)

Autosampler bottle (8×30mm, 1ml), snap cap

Safety : This method requires the use of a fume hood and involves exposure to chemicals. Review safety regulations for fume hood use and chemical spills.

Instrument model manufacturer

Automated Microlab ^® SPE Hamilton

Pump/HPLC Syringe HP 1100 Agilent

Detector RF10AXL Shimadzu

Data System Chemstation Agilent

Chromatographic column

Phenomenex Luna 100×4.6mm C-18(2)3 microns # 00D-4251-EO

Preparation of reagents

Diluent (pH 8.3 to 8.5; 1000ml).

1. Weigh 3.0 grams of sodium borate, 3.0 grams of boric acid, and 8.0 grams of sodium bicarbonate into a tared dry beaker.

2. Place an empty 800ml beaker on a magnetic stirrer. Add approximately 500ml Milli-Q ^™ water and a stir bar. Stir the water vigorously, but don't splash it out.

3. Quantitatively transfer the reagent from step 1 into water; stir until it is completely dissolved.

4. Quantitatively transfer the solution from step 3 to a 1L volumetric flask, and dilute to volume with Milli-Q ^TM water; mix well. Stable for six (6) months.

Calcium chloride solution (100 g).

1. Weigh 40 grams of calcium chloride dihydrate into a tared 250ml beaker.

2. Add 60 grams of Milli-Q ^™ water. Mix well. Store under ambient conditions in a covered glass bottle. Can be placed stably for 1 year.

Extraction solvent (pentane: ethyl acetate 80:20; 500ml)

Safety : Pentane and ethyl acetate are volatile and flammable. Perform the following operations in a fume hood.

1. Transfer 400ml of pentane to a 500ml HPLC storage bottle.

2. Add 100 ml of ethyl acetate. Mix well. Store in/under cover in a fume hood.

Mobile phase (buffer: methanol: acetonitrile 60:5:35, pH3.2, 2L)

1. Weigh 1.35 grams of tetramethylammonium chloride, 3.65 grams of citric acid and 1.60 grams of sodium citrate in a tared dry beaker.

2. Place an empty 800ml beaker on a magnetic stirrer. Add approximately 500ml Milli-Q ^™ water and a stir bar. Stir the water vigorously, but don't splash it out.

3. Quantitatively transfer the reagent from step 1 into water; stir until it is completely dissolved.

4. Quantitatively transfer the solution from step 3 to a 1L graduated cylinder, and dilute to 1000ml with Milli-Q ^TM water; mix well.

5. Transfer to a 2 liter HPLC mobile phase reservoir.

6. Add 200ml Milli-Q ^™ water, 100ml methanol and 700ml acetonitrile. With vigorous stirring, the latter two solvents were added slowly. Do this in a fume hood and wear personal protective equipment. See the relevant Material Safety Data Sheet (MSDS) for details.

7. While stirring, degas the mobile phase with vacuum suction.

FMOC reagent solution (in acetone)

1. Weigh 0.10 g of FMOC reagent into a tared 100 ml volumetric flask.

2. Add acetone to dissolve, and use it to dilute to volume. Mix well. Do this in a fume hood. Wear the PPE specified in the MSDS of the chemical.

3. Freeze for no more than six (6) months.

Acid solution (5% HCl ) for sample extraction

1. Add 100ml of Milli-Q ^TM water to a 200ml volumetric flask.

2. Add 4ml of 1N HCl to the volumetric flask.

Dilute to volume with Milli-Q ^™ water.

Prepare internal standard (aminoisobutyric acid)

ISTD A-internal standard stock solution A

1. Weigh 0.5 g of aminoisobutyric acid into a tared 250 ml volumetric flask.

2. Add 25ml 1.0N HCl and about 100ml Milli-Q ^™ water. Vortex to mix until dissolved.

Dilute to volume with Milli-Q ^TM water and mix well. Store frozen for no longer than six (6) months.

ISTD B - Working Internal Standard Solution B (Add this solution to the Calibration Standard)

1. Pipette 1ml of internal standard solution A into a 100ml volumetric flask.

2. Dilute to volume with Milli-Q ^™ water. Stable for 1 month.

Prepare Calibration Standards

Stock calibration solution .

In a tared 50ml volumetric flask, weigh 0.100g (+/-0.001g) asparagine and 0.100g (+/-0.001g) aspartic acid. Add 25ml of Milli-Q ^™ water and 1ml of 1N HCl. Place it in a sonic bath until dissolved, then dilute to volume with Milli-Q ^™ _H2O . The solution remains effective for 6 months when refrigerated.

working standard .

Prepare the following working calibration standards : Std# mL stock solution Final volume (mL) ppm 1 5 200 50 2 5 100 100 3 1 10 200 4 3 10 600

The solution remains effective for 1 month when refrigerated.

Prepare samples

1. Weigh 1g of sample into a 125ml Erlenmeyer flask.

2. Add 48.0ml of 5% HCl solution to each sample.

3. Add 2ml ISTDA A to each sample.

4. Cover each flask with aluminum foil and place in a water bath at 60C for 30 minutes.

5. 10 mL dichloroethane in each sample.

6. Homogenize the sample for 60 seconds.

7. Pour a portion of the sample into a 30ml centrifuge tube.

8. Centrifuge at 10000 rpm for 32 minutes at 5°C. The supernatant was used in the "sample-dilution" step 1.

Prepare standards and samples

Three Microlab( ^R) methods were used to dilute samples/standards, add internal standards and form FMOC derivatives. They are outlined below.

Microlab method used for operation

Dilution TRANSDIL

Add internal standard ADDISTD

Formation of FMOC derivatives ADDFMOC

Use MICROLAB ^ Automated preparation of samples and standards

Step 1: Standards - Addition of ISTD and Dilution Steps

1. Prepare two sets of tubes for each standard. In one set of tubes, place approximately 2 mL of standards, and place these tubes containing standards in the leftmost position on the ^Microlab® .

2. Place the rack containing the empty tubes in the rightmost rack position of the ^Microlab® .

3. Inject Working Internal Standard Solution B into a 20ml glass (scintillation) tube and place on the working area of the Microlab ^(R) .

4. Select the method ADDISTD. (Mix 200 μl ISTD B, 50 μl standard solution, dilute total volume to 4000 μl with Milli-Q ^™ water).

5. Implement the method.

6. Remove the tube set from the left position and set aside.

7. Remove the working internal standard solution from the Microlab ^(R) workspace and freeze. Leave the right tube for step 3.

Step 2: Sample-dilution step (ISTD has been added during sample preparation)

1. For each sample, prepare two sets of tubes. In one set of tubes, approximately 2 mL of sample was placed, and these tubes with samples were placed in the leftmost position of the ^Microlab® .

2. Place the rack containing the empty tubes in the rightmost rack position of the ^Microlab® .

3. Select method TRANSDIL. (Set the sample number, the sample volume is 50 μl, and the final dilution volume diluted with Milli-Q ^TM water is 4000 μl.)

4. Implement the method.

5. Remove the tube set from the left position and set aside.

Leave the right tube for step 3.

Step 3: Adding FMOC Reagent - Preparation of Fluorescent Derivatives

1. Prepare a 100×16mm screw cap tube.

2. Place the tube rack on the rightmost rack position of the ^Microlab® .

3. Place the standard and sample tubes from the dilution step above in the leftmost rack position of the Microlab ^(R) .

4. Transfer an aliquot (22ml) of the FMOC reagent solution to a glass scintillation vial. Add approximately 100 μL of 40% calcium chloride solution; mix well. (Calcium chloride is added to "charge" the FMOC reagent - this is required for the Microlab ^(R) assay).

5. Place the scintillation tube on the ^Microlab® workspace.

6. Select method ADDFMOC.

7. Switch syringes 1 and 2 from water to diluent (pH 8.3 to 8.5).

8. Wash syringes 1 and 2 with diluent (pH 8.3 to 8.5) for at least 5 cycles.

9. Implement method ADDFMOC. (Mix 450 μl FMOC solution, 250 μl sample from ADDISTD above, dilute with diluent solution to a final volume of 1300 μl).

10. Remove the tube set from the sample rack position and discard.

11. Remove the FMOC reagent solution from the Microlab( ^R) workspace and freeze.

13. Remove the tube set from the far right position and place in a fume hood. Let stand for at least 10 minutes, or until the solution is clear (but no longer than 20 minutes).

14. Add 2ml of extraction solvent to each tube. Cap and vortex at high speed for 2 min to extract unreacted FMOC reagent.

15. Prepare another tube holder for 55×16mm tubes. Add 1ml of mobile phase solution to each tube.

16. Transfer 1.0 mL of the aqueous layer (lower layer) from the centrifuge tube to a 55 x 16 mm tube.

17. Discard the upper layer (organic layer).

18. Transfer the sample to an autosampler vial and seal.

Chromatography

operating conditions

HP1100 with Chem Station software

Detector: Waters474 scanning fluorescence detector

Model: Norm

Signal: 0.0000

Wavelength: excitation 260

Launch 313

Gain: 10

Decay: 1

Response: FST

Chromatographic column: Phenomex Luna C18(2) 100×4.6mm 3u

LC method

Flow: 1.000ml/min

Isocratic operation (see reagent preparation - mobile phase)

Injection volume: 10.0ul

Temperature setting: no temperature control

calculate

Using the area values, calculate the sample solution against a standard curve of known quantities:

y=mx+b

y(asparagine/ISTD ratio)=m(slope)x(asparagine concentration)+b(y-intercept)

(y-b)/m=x

Asparagine (ppm) = (Asparagine Area/ISTD Area-Intercept)/Slope

[ppm=ug/mL]

Example

Asparagine (ppm) = (215.45436/551.828--0.0165)/0.0023 = 176.93ppm

Correction for dilution/homogenization during sample preparation steps.

Asparagine (ug/g) = measured asparagine (ppm) X sample diluent volume (50) (ml)

grams of sample

[ppm=ug/mL]

Example

Criteria for judging eligibility :

●The accuracy of the working reference material control sample must be within 10% of the known result for asparagine.

• The linearity (r ² ) of the calibration curve must be 0.995 or greater.

LC Chromatographic Analysis of Samples

Figure 3 lists the LC chromatogram analysis of the samples. keep time compound 4.5 minutes Asparagine 6.6 minutes aspartic acid 11.5 minutes FMOC reagent 20.7 minutes ISTDs

3. Percentage reduction of acrylamide

% reduction of acrylamide = [(acrylamide content in the control sample - acrylamide content in the treated sample)/acrylamide content in the control sample] x 100.

Control samples were prepared in exactly the same manner as the treated samples, except that the control samples had not been treated for asparagine removal.

4. Percent reduction of asparagine

Percent reduction of asparagine = [(asparagine content in control sample - asparagine content in treated sample)/asparagine content in control sample] x 100.

Control samples were prepared in exactly the same manner as the treated samples, except that the control samples had not been treated for asparagine removal.

Example

The following examples illustrate the invention but are not intended to limit it.

Example 1 - Dehydrated Potato Products Containing Reduced Amides

Peel and cut Russet potatoes into 0.64cm (”in) thick slices. Steam in water until tender (~93°C (200°F) for 30 minutes). Drain and rinse with water. The cooked slabs were passed through a wire mesh to make mashed potatoes (1.67mm center-to-center distance of the mesh wires). A portion of this mashed potato was set aside as a control mashed potato.

Add 453 g (1 lb) mashed potatoes and 1,361 g (3 lb) water to a Kitchen-Aid( ^R) food processor (model #K45) with a whisk at speed = 2 per minute. The mixture was filtered through a coarse sieve (mesh center distance 1.67 mm). The liquid portion (filtered) was filtered through a fine sieve (mesh center distance: 1.11 mm). Mix the two solid fractions. Repeat the first wash five times.

Mix all the one-wash parts together. The washing process was repeated on the first wash mashed potatoes to obtain -1,905 g (4.2 lbs) of second wash mashed potatoes.

The mashed potatoes after the second wash and the control mashed potatoes were dried with a freeze dryer.

The asparagine content (on a dry weight basis) of the control mashed potatoes and the post-washed mashed potatoes was 1.2% and less than 0.2%, respectively. The reduction of asparagine by this method was greater than 84%.

Example 2 - Processed Potato Snack Products Containing Reduced Amides

The control and twice washed dehydrated potato products of Example 1 above were used to make processed potato snacks as described in the table below: Control after the second wash Dehydrated Potato Products Control 100g Dehydrated potato products after secondary washing 100g Emulsifier 0.6g 0.6g water 50.5g 50.5g frying temperature 191°C (375°F) 191°C (375°F) frying time 8 seconds 8 seconds

Grind the dried potato products into powder. Mix water and emulsifier, then mix thoroughly with flour to form dough. The dough is then passed through grinding rollers to form sheets, and the sheet-shaped dough is then cut into oval dim sum pieces. The dough is then fried in hot oil to form a processed potato snack.

The moisture and acrylamide contents of the two samples are listed in the table below: Final product Control 2X after washing Moisture (%) 1.8 1.1 Acrylamide(ppb) 3219 60

The processed potato snacks using the extraction method of Example 1 had a greater than 98% reduction in acrylamide.

Example 3 - Potato Chips

Blanched only : Using raw potato chips, it is possible to make potato chips with reduced acrylamide content. Atlantic potatoes were peeled and cut into ~1.1 mm thick slices. Wash, and press dry. Blanch the potato slices in 74°C (165°F) water for 15 seconds. Let cool and drain water from blanched potato chips. The treated potato chips were fried in a deep fryer set at 191°C (375°F) for 60 seconds. (Example 2A) Two batches of potato chips were made as above, except that the blanching time was changed to 60 seconds (Example 2B) and 180 seconds (Example 2C).

A control sample was prepared in the same manner as used to prepare Examples 2A, B and C above, except that the potato flakes were not blanched.

Using the method set forth herein, the samples and controls were analyzed for acrylamide content. hot only 2A 2B 2C Blanching time (seconds) 15 60 180 Acrylamide in control sample ^* (ppb) 1079 1079 1079 Acrylamide in blanched samples (ppb) 940 696 679 Percentage reduction (%) 13 35 37

^* Note - only one control sample was prepared

Blanched and Extracted : Using raw potato chips, chips with reduced acrylamide can be made. Atlantic potatoes were peeled and cut into ~1.1 mm thick slices. Wash, and press dry. Blanch the potato slices in 74°C (165°F) water for 15 seconds. Let cool and drain water from blanched potato chips. It was extracted by soaking 100 g of blanched potato slices in 250 ml of distilled/deionized water for one hour. Vortex samples for 1 min every 8 min. Heat in a microwave oven (Panasonic microwave oven, model NN-S5488A) set to high heat for 2 minutes, and then wash with about 800ml of cold tap water three times for 10 seconds each time. The treated potato chips were fried in a deep fryer set at 191°C (375°F) for 60 seconds. (Example 2D) Two batches of potato chips were made as above, except that the blanching time was changed to 60 seconds (Example 2E) and 180 seconds (Example 2F).

A control sample was prepared in the same manner as used to prepare Examples 2D, E and F above, except that the potato flakes were neither blanched nor extracted.

Using the method set forth herein, the samples and controls were analyzed for acrylamide content. Blanched and extracted: 2D 2E 2F Blanching time (seconds) 15 60 180 Acrylamide in control sample ^* (ppb) 1079 1079 1079 Acrylamide in blanched and extracted samples (ppb) 281 156 50 Percentage reduction (%) 74 86 95

^* Note - only one control sample was prepared

Conventional potato chips do not undergo a blanching step, thus potato chips with a blanching step are not known in the art. The above examples demonstrate that excellent results can be obtained by using the novel combination of blanching and extraction in the potato chip manufacturing process.

Example 4 - French Fries

French fries with reduced acrylamide can be made by using raw potato strips.

Atlantic potatoes were peeled and cut into potato strips with a cross-sectional area of approximately 8mm x 8mm. Wash, and press dry. Blanch potato strips in 74°C (165°F) water for 1 minute. Let cool and drain the blanched potato chips. It was extracted by soaking 100 grams of blanched potato strips in 250 ml of distilled/deionized water for one hour. Vortex samples for 1 min every 8 min. Heat in a microwave oven (Panasonic microwave oven, model NN-S5488A) set to high heat for 2 minutes, and then wash with about 800ml of cold tap water three times for 10 seconds each time. The treated potato strips were fried in a deep fryer set at 191°C (375°F) for 3 minutes. (Example 3A) Two more batches of French fries were made as above, except that the blanching time was changed to 3.5 minutes (Example 3B) and 7 minutes (Example 3C).

Control samples were made in the same manner as used to make Examples 3A, B and C above, except that they were not extracted.

Using the method set forth herein, the samples and controls were analyzed for acrylamide content. 3a 3b 3C Blanching time (minutes) 1 3.5 7 Acrylamide in control sample ^* (ppb) 216 216 216 Acrylamide in blanched and extracted samples (ppb) 130 84 5.7 Percentage reduction 40% 61% 97.4%

^* Note - only one control sample was prepared

Example 5 - Merchandise

The potato chips of Example 3 were packaged in bags for sale to consumers. A message is printed on the bag stating "Acrylamide-Free Product!"

Example 6 - Merchandise

The potato chips of Example 3 were packaged in bags for sale to consumers. A message is printed on the bag stating "Low in acrylamide!"

Example 7 - Merchandise

The potato chips of Example 3 were packaged in bags for sale to consumers. A message is printed on the bag, stating, "Over 90% less acrylamide!" The message in the TV ad for the chips is, "Our chips are lower in acrylamide!"

Example 8 - Merchandise

The uniformly shaped processed potato chips of Example 2 having an acrylamide content of less than 100 ppb were packaged in cylindrical cans for sale to consumers. The message in the TV ad for the chips is, "Reduced acrylamide!"

Example 9 - Merchandise

The french fries of Example 4 were packaged for sale to consumers in paper sleeves that were open at one end from which the french fries protruded. "Our french fries contain a reduced amount of acrylamide!" reads a billboard posted inside a retail establishment that sells french fries!

Example 10 - Merchandise

The french fries of Example 4 were packaged for sale to consumers in paper sleeves that were open at one end from which the french fries protruded. "Our french fries are low in acrylamide!" reads a billboard posted inside a retail establishment that sells french fries!

Example 11 - Merchandise

The potato chips of Example 3 were packaged in bags for sale to consumers. A message is printed on the bag, stating "Made with ingredients low in asparagine!"

Example 12 - Dehydrated Potato Products

Wash the russet baked potatoes with water and place in a pot of boiling water. Boil (submerge) the potatoes for 1 hour. Remove the cooked potatoes from the water, peel the skins, and mash the pulp. Add 45g of water to 15g of mashed potatoes and homogenize the mixture until homogeneous and free from lumps.

200 units of glutaminase were added to the homogenized solution, and the sample was shaken every 5 minutes for 30 minutes.

After a 30 minute incubation, the product was heated in a high microwave (Panasonic Microwave Oven, model NN-S5488A) in 2 minute increments for 10 minutes until dry (and browned). When analyzed for acrylamide content by the method described herein, dehydrated potato products treated with enzymes achieved greater than 10% acrylamide compared to dehydrated potatoes prepared using exactly the same method except that no enzyme was added (control sample) Amide reduction.

While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made by those skilled in the art without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

Claims (14)

1. A method for reducing asparagine content in food materials, said method comprising: (1) providing food materials containing asparagine; (2) optionally reducing the particle size of the food material; (3) optionally increasing the cell membrane permeability of the food material; and (4) extracting asparagine from the food material. 2. The method of claim 1, wherein the cell membrane of the food material is increased by blanching the food material. 3. A method for reducing acrylamide content in food, said method comprising: (1) providing food materials containing asparagine; (2) optionally reducing the particle size of the food material; (3) optionally increasing the cell membrane permeability of the food material; (4) extracting part of said asparagine from said food material; and (5) heating the food material to form a final food product. 4. The method of claim 3, wherein the cell membrane of the food material is increased by blanching the food material. 5. A commodity comprising: (a) snack chips, wherein the snack chips contain a reduced amount of acrylamide; (b) a container containing said snack pieces; and (c) a reminder message attached to the container; Wherein the reminder message attached to the container informs the consumer that the snack chip contains a reduced amount of acrylamide. 6. The commodity of claim 5, wherein the snack chips are acrylamide containing Potato flakes in an amount less than about 40 ppb. 7. The article of manufacture of claim 6, wherein said potato chips have an acrylamide content of less than about 10 ppb. 8. The article of manufacture of claim 5, wherein the snack chips are processed potato chips having an acrylamide content of less than about 400 ppb. 9. The article of manufacture of claim 8, wherein the processed potato chips have an acrylamide content of less than about 200 ppb. 10. The article of manufacture of claim 5, wherein the snack chips are nacho chips having an acrylamide content of less than about 75 ppb. 11. The article of manufacture of claim 10, wherein the tortilla chips have an acrylamide content of less than about 50 ppb. 12. A commodity comprising: (a) French fries, wherein the French fries contain reduced amounts of acrylamide; (b) containers containing said French fries; and (c) a reminder message attached to the container; Wherein the prompt message attached to the container informs the consumer that the French fries contain a reduced amount of acrylamide. 13. The article of manufacture of claim 12, wherein the French fries have an acrylamide content of less than about 40 ppb. 14. The article of manufacture of claim 13, wherein the French fries have an acrylamide content of less than about 10 ppb.

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