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Title:
METHOD AND COMPOSITION TO REDUCE THE FORMATION OF ACRYLAMIDE IN FRESH OR PRE-FRIED FOODS TO BE SUBJECTED TO HEAT TREATMENT
Document Type and Number:
WIPO Patent Application WO/2016/001894
Kind Code:
A1
Abstract:
An enzymatic composition comprising a) L- asparaginase and b) a buffering agent as technological adjuvant for reducing the amount of acrylamide in the preparation of fresh foods and/or pre-fried and frozen foods that are to be fried or baked.

Inventors:
BELLOMARIA, Alessia (Via Romolo Balzani 85, Roma, I-00177, IT)
PACI, Maurizio (Via Corvisieri 4, Roma, I-00162, IT)
Application Number:
IB2015/055038
Publication Date:
January 07, 2016
Filing Date:
July 03, 2015
Export Citation:
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Assignee:
WEST SYSTEMS SRL (Viale Donato Giannotti 24, Firenze, I-50126, IT)
International Classes:
A23L3/3571; A21D8/04; A23B7/155
Domestic Patent References:
WO2003095496A12003-11-20
WO2007077545A12007-07-12
WO2004026042A12004-04-01
WO2004030468A22004-04-15
WO2004030468A22004-04-15
WO2004032648A12004-04-22
WO2008110513A12008-09-18
WO2008151807A22008-12-18
WO2010070010A12010-06-24
WO2009152348A12009-12-17
WO2004028278A22004-04-08
Foreign References:
US20110256267A12011-10-20
US20070141225A12007-06-21
US20100040729A12010-02-18
CN102138909A2011-08-03
US20090047725A12009-02-19
GB1313006A1973-04-11
US3652402A1972-03-28
EP1715757A12006-11-02
US20120128828A12012-05-24
US20130023029A12013-01-24
US20110256267A12011-10-20
US20070141225A12007-06-21
US20100040729A12010-02-18
US20060083832A12006-04-20
US20040105929A12004-06-03
US20040115321A12004-06-17
US20030219518A12003-11-27
US20070196556A12007-08-23
US7396670B22008-07-08
US20040058045A12004-03-25
US20100255167A12010-10-07
US20040109926A12004-06-10
Other References:
AMREIN T. M. ET AL.: "Acrylamide in gingerbread: Critical factors for formation and possible ways for reduction", J AGRIC FOOD CHEM, vol. 52, 2004, pages 4282 - 4288, XP002613725, DOI: doi:10.1021/JF049648B
ZYZAK D. V. ET AL.: "Acrylamide formation mechanism in heated foods", J AGRIC FOOD CHEM, vol. 51, 2003, pages 4782 - 4787, XP002276619, DOI: doi:10.1021/jf034180i
TAUBERT D. ET AL.: "Influence of processing parameters on acrylamide formation during frying of potatoes", J AGRIC FOOD CHEM, vol. 52, 2004, pages 2735 - 2739
LINDSAY R. C.; JANG S.: "Chemistry and Safety of Acrylamide in Food", 2005, SPRINGER, article "Chemical intervention strategies for substantial suppression of acrylamide formation in fried potato products", pages: 393 - 404, XP008163375
RYDBERG P. ET AL.: "Factors that influence the acrylamide content of heated foods. Chemistry and Safety of Acrylamide in Food", 2005, SPRINGER SCIENCE, pages: 317 - 328
LOW M. Y. ET AL.: "Effect of Citric Acid and Glycine Addition on Acrylamide and Flavor in a Potato Model System", J AGRIC FOOD CHEM, vol. 54, 2006, pages 5976 - 5983
MESTDAGH F. ET AL.: "Impact of additives to lower the formation of acrylamide in a potato model system through pH reduction and other mechanisms", FOOD CHEM, vol. 107, 2008, pages 26 - 31, XP022322321, DOI: doi:10.1016/j.foodchem.2007.07.013
FRIEDMAN M. ET AL.: "Review of methods for the reduction of dietary content & toxicity of acrylamide", J AGRIC FOOD CHEM, vol. 56, 2008, pages 6113 - 6140, XP008162867, DOI: doi:10.1021/jf0730486
Attorney, Agent or Firm:
FEZZARDI, Antonio et al. (Via Collina 36, Rome, I-00187, IT)
Download PDF:
Claims:
CLAIMS

1) An enzymatic composition for reducing the formation of acrylamide in fresh or pre-fried foods to be subjected to thermal treatment, comprising:

a) L-asparaginase ; and

b) sodium bicarbonate,

in aqueous solution with pH comprised between 6 and 7, at room temperature, in a volume such as to wet the food to be treated completely.

2) The enzymatic composition according to Claim 1, wherein L-asparaginase and sodium bicarbonate are in a quantitative weight ratio of 1 to 1200-1600.

3) The enzymatic composition according to Claims 1 and 2, comprising 0.5 mg of L-asparaginase and 700 mg of sodium bicarbonate in a volume of approximately 300 ml of enzymatic aqueous solution at room temperature effective for the treatment of a kilogram of fresh or pre-fried food.

4) The enzymatic composition according to Claims 1 to 3, further comprising food additives with thickening, gelling, and stabilizing function.

5) The enzymatic composition according to Claim 4, comprising microcrystalline cellulose.

6) Use of the enzymatic composition comprising L-asparaginase, sodium bicarbonate, and food additives as technological adjuvant for reducing the amount of acrylamide in the preparation of fresh and/or pre-fried foods, and/or pre-fried and frozen foods, that are to be fried or baked.

7) A method for reducing the amount of acrylamide in fresh foods and/or pre-fried and frozen foods in the preparation of a food product to be fried or baked, comprising the steps of:

- dissolving an amount of enzymatic composition comprising L-asparaginase , sodium bicarbonate, and appropriate food additives, in a volume of running water, or, in the case of a frozen product, in its thawing water, such as to wet the amount of food to be treated completely;

- wet completely, by dipping or sprinkling, the vegetables reduced into sizes suitable for the subsequent cooking stage with the enzymatic composition in aqueous solution at room temperature for approximately 20 minutes;

- removing the aqueous solution, possibly further washing the vegetables rapidly in water, and drying them;

- frying the vegetables or subjecting them to thermal treatment for cooking.

8) The method according to Claim 7, wherein the food derives from a raw material of vegetable origin with high starch content.

9) The method according to Claim 7 and 8, wherein the vegetable food is potato, sweet potato, tapioca, yam, carrot, turnip, or parsnip.

10) Food obtained by means of the method according to Claims 7 to 9.

Description:
METHOD AND COMPOSITION FOR REDUCING THE FORMATION OF ACRYLAMIDE IN FRESH OR PRE—FRIED FOODS TO BE SUBJECTED TO HEAT TREATMENT

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FIELD OF APPLICATION OF THE INVENTION

The present invention relates to the food sector and, more precisely, regards a method for reducing the formation of acrylamide in foods, either fresh or pre- fried, to be subjected to cooking by being fried or baked, and for producing foods that have considerably reduced levels of acrylamide. The method and the product obtained therewith can be used in the domestic environment, but their use can be extended also to the food industry of the large-scale retail trade.

PRIOR ART

In the food industry, great interest at a world level has led to extensive research carried out on the toxic effects that have emerged associated to acrylamide exposure, such as neurotoxicity, reproductive toxicity, genotoxicity, clastogenicity , and cancerogenicity . Acrylamide was classified in 1994 by the International Agency for Research on Cancer (IARC) as probable class II carcinogenic compounds for humankind. So far multiple pathways for the formation of acrylamide have been described. The main one is the Maillard reaction, where reducing sugars react with the amino acid asparagine at temperatures higher than 120 °C in a condensation reaction to form acrylamide. Among the various foods that have proven most concerned, one of the most important is represented by potatoes in the form of fried chips, crisps, and French fries since they contain a considerable amount of free asparagine.

The various studies conducted show that multiple factors can affect formation of acrylamide, amongst which the type of potato, the system of cultivation, the harvesting time, and the storage time and temperature. Furthermore, the production of acrylamide can be affected by various parameters that are strictly related to the Maillard reaction, i.e., the concentration of the precursors of the reaction (sugars and amino acids) , the presence of water in the system, and the possibility of using additives, such as antioxidants .

In the last decade, various methods have been devised and patented for reducing the formation of acrylamide. These methods act principally on parameters that regard the various operating steps of preparation of fried foods. For instance, tests have been conducted on vacuum frying or conventional frying (at atmospheric pressure) at low temperature [1-6] . Furthermore, post- frying techniques have been developed for the removal of acrylamide. However, their use has proven impracticable since it destroys the microstructure of the foods and alters the nutritional properties thereof .

One of the most promising and advantageous strategies for reducing sensibly the content of acrylamide in foods cooked at high temperatures consists in intervening on the kinetics of the reaction that leads to formation of acrylamide, for example by reducing, prior to cooking, the amount of its precursors in the raw materials represented, that is, by reducing sugars and asparagine.

Various methods have been developed that exploit biological reactions, including the use of enzymes or the addition of amino acids [7-10] . Also preventive operations, like soaking the raw material in a solution of sodium chloride, and procedures of removal after cooking have been studied.

The effect of temperature, heating time, surface- to-volume ratio, and level of browning are factors that affect in a determining way formation of acrylamide in fried potatoes and has been amply studied by Taubert et al. It has been noted, in fact, that in forms of potatoes with low surface-to-volume ratio, the formation of acrylamide increases proportionally to the increase in temperature and the processing time. On the other hand, in forms with intermediate or high surface- to-volume ratio, the maximum formation of acrylamide occurs at temperatures of between 160-180°C. Furthermore, studies have been carried out regarding the effect of water on formation of acrylamide, with the conclusion that by controlling humidity, it is possible to reduce partially secondary reactions of the Maillard reaction [11] . Also important is the effect of metal ions in the formation of acrylamide. As various studies have shown, the use of NaCl and CaCl 2 , for example, could minimize formation of acrylamide during frying, suggesting that the ionic and electrostatic interactions between cations and asparagine suppress the Maillard reactions in the initial phase, probably following upon polymerization reactions [12] . The preventive effect of Ca 2+ ions may be due also to the inhibition of the reactions that involve Schiff base, which is an intermediate of the Maillard reaction [13] .

In 2010, Bourg et al . disclosed a method for reducing the level of acrylamide in foods containing starch, such as maize, wheat, barley, rye, and mixtures thereof. Acrylamide can be removed by polymerization, solubilization, and/or vaporization at vacuum pressure [14] .

Rydberg et al . studied the effect of pH on the formation of acrylamide, concluding that the formation of acrylamide has a maximum around pH values of 8. Lower pH values determine a slowing-down or total prevention of its production [15] . The effect of the reduction of pH can be obtained by addition of organic acids (benzoic acid, propionic acid, and sorbic acid) or of citric acid and the amino acid glycine [16] . However, the low pH can adversely affect the taste of the foods according to the concentration of organic acids added [17] .

After various attempts aimed at removal of acrylamide in the food after its formation, the strategy of preventing formation thereof has been pursued, with the reduction of the amount of its precursors in the raw materials. In this sense, the various pre-treatments patented regard the addition of micro-organisms such as yeasts, bacteria, and fungi [18], or the addition of enzymes, first of all asparaginase, the enzyme that converts free asparagine into aspartic acid, which, thus removed, cannot participate in the Maillard reaction and hence cannot contribute to formation of acrylamide [19] . Use of asparaginase that hydrolyses the asparagine to aspartic acid may prove very effective for reducing formation of the acrylamide that is formed, through the Maillard reaction, when foods containing starch are baked or fried. The reaction is responsible, among other things, for browning of these foods and their toasted fragrance.

The asparaginase enzyme is hence used as technological adjuvant for reducing the levels of L- asparagine.

It is well known, in fact, that the formation of acrylamide in heated or cooked foodstuff products can be reduced with a treatment designed to reduce the amount of asparagine in the food by subjecting the food to the action of the asparaginase enzyme, as described in the patent application No. WO2004/026042. By adding asparaginase prior to cooking of the food, asparagine is converted into aspartic acid and ammonium. As a result, asparagine cannot take part in the Maillard reaction, and formation of acrylamide is considerably reduced, even though it is impossible to prevent it altogether. On average, asparaginase reduces formation of acrylamide by 90%, without the foods undergoing modifications of taste or appearance.

To adapt the treatment with asparaginase to the line for industrial production of a food product, it should preferably be carried out during one of the steps of the production process. However, it is problematical to insert the treatment with asparaginase in existing industrial-production processes, for example for fried chips or crisps, because the enzyme is not stable and does not maintain its activity at the temperatures of the various typical steps of the process .

Recent technological applications use asparaginase by adding it to the food prior to the heating step, precisely to prevent, as a result of heating, the enzyme from being denatured and hence deactivated. The international patent application No. WO 04/030468 provides a method for preventing formation of acrylamide by treating an intermediate form or precursor of a food, or food product, with an enzyme that breaks down the amino acids involved in formation of acrylamide. The method comprises the addition of one or more enzymes that are able to modify the amino acids that participate in the formation of acrylamide, on the surface of the foodstuff precursor, and a subsequent heating step. However, in some cases said method could be difficult to implement, for example in the case where the content of asparagine is rather high, precisely as in the case of products deriving from potatoes. Considerably high levels of asparagine could require too long processing times that could jeopardize the quality of the end product.

According to the teaching of the European patent application No. EP 1 715 757 it is not necessary to remove or convert all the amino acids or the majority of the amino acids present in the food, but it is sufficient to remove or convert those present in a thin surface layer of the food. The preferred enzyme for executing this process is an enzyme active on asparagine and glutamine; also forming part of the method is the step of heating the food to a temperature of 100°C or higher. A further advantage of this method would consist in the fact that the food product thus obtained contains low amounts of acrylamide but high amounts of asparagine or glutamine.

The U.S. patent application No. US 2012/0128828 describes the synergistic action of asparaginase and at least one hydrolysing enzyme in an enzymatic composition designed to reduce acrylamide in the foods thus treated.

Asparaginase is produced with recombinant-DNA techniques and is purified from bacterial culture. For commercial purposes, the enzymatic concentrate is either a liquid or granular product on the basis of the foodstuff category in which it is to be used.

There have also been developed more innovative procedures for producing the enzyme, or its alternative forms with specific characteristics, which involve genetic-engineering technologies that envisage modification of vegetable DNA [20] .

Even though all the L-asparaginases catalyse the same chemical conversion, they do not all have the properties suitable for the same application. Various applications have different requirements as regards the conditions in which the enzyme must be able to act. It is known that the physical and chemical parameters that can affect the kinetics of the enzymatic conversion are temperature, which has a positive effect on the rate of the chemical reaction but a negative effect on the stability of the enzyme, humidity, pH, saline concentration, structural integrity of the food, presence of enzyme activators and inhibitors, concentration of the substrate and of the product, etc.

Thus, asparaginases specifically suitable for the purpose have been isolated from different organisms belonging to the kingdom of the fungi, for example Aspergillus niger as described in the document No. W02004 / 030468 and Aspergillus oryzae described in the document No. W02004 / 032648.

The document No. W02008 / 110513 describes thermostable variants of asparaginases obtained from Aspergillus oryzae. One of the variants produced enables the treatment of chips, crisps, or French fries for 5 minutes at 60°C, but this temperature is not ideal for industrial use on account of the potential microbial growth at the above temperature.

Also the use of hyperthermostable asparaginases described in the document No. W02008 / 151807 , albeit suitable for being applied in the steps of the industrial process at high temperature, is not optimal since these enzymatic forms are in turn inactive in the process steps that are carried out at lower temperatures .

The technical solution proposed by the invention forming the subject of the patent application No. W02010 / 070010 consists in setting the chips, crisps, or French fries in contact with asparaginase at a temperature of 65°C in the presence of sodium acid pyrophosphate. But also the temperature of 65°C is not sufficiently high to counter the risk of microbial contamination.

New polypeptide variants of asparaginase, the corresponding encoding polynucleotide sequences, and their uses form the subject of the patent application No. US2013 / 0023029. As a result of appropriate genetic variations in the nucleotide sequence encoding asparaginase there is a widening of the pH activity profile, i.e., of the width of the range of pH in which the enzyme has its maximum activity.

However, recourse to enzymatic variants of asparaginase that may be used in any industrial process, produced with complex genetic-engineering technologies, renders the solution an approach that is far from practicable and very costly given the amounts of enzyme necessary for treating the amounts of fried chips or crisps consumed all over the world.

The U.S. patent application No. US 2011/256267 describes a composition comprising asparaginase and a buffering agent, for example citric acid and the like, at pH 6-7 for reducing formation of acrylamide in foods, in particular potatoes, chips, crisps and the like, and oven baked products, use of which envisages that the composition is sprayed on the food, and this is followed by baking or frying.

Also the U.S. patent application No. US 2007/141225 describes a composition comprising asparaginase and an acid buffering agent at pH 6-7 that is able to reduce formation of acrylamide in foods like potatoes, chips, or the like, and oven-baked products. The composition is sprayed on the food prior to baking or frying.

The U.S. patent application No. US 2010/040729 likewise describes a composition comprising asparaginase and citric acid, at pH 6-7, which is useful for reducing the formation of acrylamide in foods and is sprayed on the food prior to baking or frying .

Consequently, the teaching of the prior art is to use citric acid as buffering agent to provide the conditions of reaction ideal for the activity of asparaginase .

From what has been described so far, it is evident that, notwithstanding the numerous studies conducted to implement the reaction of abatement of acrylamide by the action of asparaginase, the results obtained so far are not very satisfactory, and the methodology of application is still under definition. There is consequently still felt the pressing need for improved and simpler methods for reducing the content of acrylamide in foods and foodstuff products rich in starch that use asparaginase enzyme.

SUMMARY OF THE INVENTION

A first purpose of the present invention is to provide a simple and economically advantageous method that can be applied in the domestic environment, but may also be extended to the industrial level, for the production of fresh foods that are to undergo frying and/or pre-fried foods that are to undergo second frying and/or baking that will be able to reduce formation of acrylamide up to 80%, using as technological adjuvant a composition comprising an appropriate amount of asparaginase.

Another purpose of the present invention is to provide a method for preventing physiological accumulation of acrylamide in consumers of foods, principally of foodstuff products containing starch, following upon processes of cooking at a high temperature, such as frying of foods or second frying of pre-fried foods.

This has been achieved by creating an additive comprising an appropriate amount of the asparaginase enzyme to be used for washing vegetables that are rich in starch and fresh and/or pre-fried vegetables in aqueous solution, prior to cooking.

Further characteristics and advantages of the invention will emerge clearly from the ensuing description with reference to the attached plate of drawings .

DESCRIPTION OF DRAWINGS

Figure 1 shows the chromatograms obtained by RP- HPLC analysis of the filtered aqueous phase in which potatoes have been left to soak for 20 minutes with the composition according to the invention and then fried; namely,

(A) treatment without asparaginase;

(B) treatment with asparaginase; and

(C) superposition of the chromatograms (A) and (B) .

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an enzymatic composition comprising asparaginase and a buffering agent to be used as technological adjuvant for the preparation of fresh foods, and/or pre-fried foods, and/or pre-fried and frozen foods, to be fried or baked . By the term "technological adjuvant" is meant herein a substance used in the food-processing transformation industry to obtain the food from the raw material or else to obtain foodstuff products with better organoleptic characteristics. Technological adjuvants differ from additives in so far as, once the desired effect has been obtained, they are taken away from the food.

The asparaginase used in the enzymatic composition according to the present invention is an L-asparaginase produced from a modified strain of Aspergillus niger, which contains the asparaginase gene of A. niger. The enzyme is isolated from the fermentation broth by filtration and concentrated by ultrafiltration. The enzyme, which has a primary structure of 378 amino acids, is a glycoprotein with an apparent molecular mass of approximately 50 kDa and an isoelectric point of approximately 3.6, is active between 20 and 60°C, and has an optimal temperature of around 25°C at pH = 7. The enzyme is rapidly deactivated when the temperature is brought to 70°C. Its activity is practically unaltered in the presence of salt.

According to the present invention, the enzymatic composition comprises L-asparaginase and a buffering agent, and may further comprise appropriate food additives with thickening, gelling, and stabilizing functions, for example microcrystalline cellulose.

The buffering agent according to the present invention is an additive that is able to stabilize the pH at a moderately acidic value of between 6 and 7, without altering the organoleptic properties of the food. Furthermore, this buffering agent must be very soluble in water, and must be removable rapidly by washing in running water.

A variety of buffering systems known to the operators of the food-industry sector may be used in some embodiments according to the present invention, such as for example citric acid, ascorbic acid, tartaric acid and their salts, bicarbonate salts, monobasic/dibasic sodium-phosphate system.

It has been found that sodium bicarbonate provides the buffering capacity adequate to maintain the pH value in the range necessary for optimal activity of asparaginase without altering the final taste of the food. In fact, unlike other buffering systems, and in particular citric acid, preferably used in the methods described in the documents of the prior art closest to the present invention, bicarbonate and sodium ions are in themselves already present in running water or in the thawing water of frozen foods, which according to the invention provide the solvent in which the enzymatic composition comprising asparaginase and buffering agent is dissolved. Furthermore, the bicarbonate ion and the sodium ion are completely odourless, colourless, and tasteless. Consequently, unlike citric acid, which, even at a residual level in the amount of aqueous solution comprising the enzymatic composition that remains on the food after treatment, leaves on the food a tart and slightly acid taste, said buffering system leaves the organoleptic properties of the food completely unaltered.

Furthermore, according to the invention, after the enzymatic treatment, there is envisaged removal of the aqueous solution comprising the enzymatic composition, possibly carrying out a further rapid washing in water and drying of the vegetables, and a possible further step of rinsing in order to remove as much as possible any contamination of enzymatic composition and sodium bicarbonate on the end food product, keeping its properties unaltered.

Hence, a composition comprising asparaginase and sodium bicarbonate, within a certain quantitative ratio, and within certain concentration ranges, which depend upon the amount of food to be treated in an adequate volume of aqueous solution, is able to offer the best performance in terms of reduction of acrylamide in the subsequent frying/cooking stage and of preservation of the original taste of the food.

There has thus been identified the amount of sodium bicarbonate and the ratio with the amount of enzyme such as to stabilize the pH value of the aqueous solution in which the enzymatic reaction occurs in the range between 6 and 7, which guarantees the maximum enzymatic activity at room temperature in a period of approximately 10-20 minutes.

In a particularly preferred embodiment of the invention, L-asparaginase and sodium bicarbonate are present in the composition forming the subject of the invention in a weight ratio of 1:1200-1600.

In an embodiment of the invention that envisages cooking of fresh food, the amount of enzymatic composition according to the invention, adequate for treatment of 1 kg of food, is previously dissolved in an amount of mains water of 300 ml. The aqueous solution thus prepared comprises 0.5 mg of L- asparaginase (150 ASPU) and 700 mg of sodium bicarbonate, and possibly microcrystalline cellulose may also be present.

Treatment of fresh foods and/or pre-fried and frozen foods, which are to be fried or baked, with the enzymatic composition according to the invention enables abatement of the production of acrylamide in the subsequent frying and/or cooking step up to 80%.

The fresh foods and/or pre-fried and frozen foods to be fried or baked that may undergo the treatment with the composition according to the invention derive from raw materials of vegetable origin with high starch content, amongst which - without this on the other hand implying any limitation - potato, sweet potato, tapioca, yam, carrot, turnip, parsnip, etc.

The fresh vegetables, washed, peeled, and cut up according to normal domestic cooking procedures so that they are reduced to pieces of dimensions suitable for subsequent frying or baking, or the products still frozen, in the case where an industrially prepared and pre-fried product is used, undergo a treatment of washing with the enzymatic composition according to the invention to reduce the amount of acrylamide in the subsequent step of domestic cooking or in the catering sector .

Consequently, a further object of the present invention consists in a method for reducing the amount of acrylamide in fresh foods and/or pre-fried and frozen foods that are to be fried or baked, comprising the steps of:

- dissolving an adequate amount of enzymatic composition comprising asparaginase, buffering agent, and appropriate food additives, in a volume of running water, or, in the case of a frozen product, in its thawing water, such as to wet the amount of food to be treated completely;

- wetting completely, by dipping or sprinkling, the vegetables reduced into sizes suitable for the subsequent cooking stage with the enzymatic composition in aqueous solution at room temperature for approximately 20 minutes;

- removing the aqueous solution, possibly carrying out a further rapid washing in water, and drying of the vegetables;

- frying the vegetables or subjecting them to thermal treatment for cooking.

In the case of treatment of industrially prepared vegetables pre-fried and then frozen, the composition according to the invention can be used also as such on the food, without being previously dissolved in water, the water of solution in this case being provided by thawing, and no further water is required.

After removal of the composition, drying and frying or cooking, according to the normal methods adopted in the home environment or in the catering sector, the product can be consumed.

Since the amount of enzyme and of buffering agent according to the invention are balanced in such a way as to guarantee the pH value at which the enzymatic activity is maximum at room temperature in aqueous solution in mains water, the invention also envisages the possibility of preparing the composition comprising L-asparaginase , buffering agent, and additives packaged in pre-packaged doses in sachets suitable for treatment of a given amount of fresh vegetable foods, or pre- fried and frozen vegetable foods, of example 1 kg, to be solubilized in 300 ml of mains water prior to cooking .

The approach adopted according to the present invention involves the use of asparaginase, which is added to the food to reduce the concentration of asparagine. By converting the asparagine into aspartic acid, the former is subtracted from the Maillard reaction that leads to formation of acrylamide. Unlike other approaches of the prior art that envisage use of the enzyme prior to cooking and then its denaturing following upon the high cooking temperatures, without removal, the present invention consists, instead, in a rapid washing of the food with the enzyme in conditions of pH that favour its maximum activity in aqueous solution with mains water in a short time at room temperature, which is easily removed through a simple washing action, is easy to apply, and involves only a short time, either at an industrial level or at a domestic level.

The results achieved with application of this method show a good acrylamide abatement. Furthermore, the enzyme, which is extremely soluble in water, is completely removed prior to frying or cooking, together with the washing water; the fresh or pre-fried food, for example potatoes, prior to frying, can also be rinsed and dried. This treatment does not modify the composition of the product, as may be evinced from the chromatograms obtained, which present a very similar pattern except for the acrylamide peak. Furthermore, the treatment according to the invention does not modify the organoleptic properties of the potatoes since all the traces of the treatment are removed with washing .

The composition according to the invention may also be used for preparing a product packaged in sachets containing the amount of composition suitable for treating a given amount of raw vegetable material suitable for domestic use, or in jars containing larger amounts, more suitable for industrial use. In this way, the treatment could be carried out prior to frying, and an end product already with a low content of acrylamide could be put on the market .

Thus, the solution proposed by the invention to the problem of accumulation of acrylamide in certain foods that are widely consumed in the population is easy to use and inexpensive.

EXPERIMENTAL PART

The invention will now be illustrated with reference to non-limiting examples of formulation, modalities of use, and test assays described hereinafter .

The aim of the work was to find the best conditions for effective application of the asparaginase enzyme in the abatement of formation of acrylamide in particular in potatoes, which are the raw vegetable material with highest content of asparagine, which is a precursor of acrylamide. From the preliminary analyses carried out on commercially produced fried chips or crisps, a method was provided for detecting a chromatographic peak corresponding to the acrylamide present using HPLC as analytic technique .

The next step was to develop the conditions of action of the enzyme, i.e., the minimum amount that can be used for achieving enzymatic saturation, as well as the shortest time necessary for its action. Various tests were conducted, assessing the applicability of different conditions of reaction in order to evaluate the results obtained in the various conditions. After analysing the data obtained from the various tests, a protocol was drawn up for the reaction of abatement, which enables use of the smallest amount of enzyme necessary and the shortest treatment time. The reaction was principally characterized for two types of potatoes, fresh ones and pre-fried and frozen ones. Example 1 - Enzymatic treatment of specimens of fresh potatoes

50 g of potatoes were cut up into cubes with a side of a few centimetres (approximately 2 cm) .

The potato cubes were soaked in approximately 200 ml of mains water, in which 0.2 mg of asparaginase (0.2 mg, 60 ASPU, an amount that brings the enzymatic kinetics to saturation), 300 mg of sodium bicarbonate, and approximately 1 g of microcrystalline cellulose were dissolved. The pH value of the solution in which the potatoes were soaked was 6.9.

The potatoes were left to soak for 10 minutes at room temperature (25°C) .

Then the washing water was removed, and the potatoes were dried and fried.

As control, the same procedure was carried out on an identical specimen of potatoes treated, in the conditions described, only with microcrystalline cellulose .

Each test was repeated in triplicate.

Example 2 - Enzymatic treatment of specimens of pre- fried and frozen potatoes

50 g of pre-fried and frozen potatoes were set in contact and mixed with a mixture constituted by 0.2 mg of asparaginase (0.2 mg, 60 ASP, a quantity that that brings the enzymatic kinetics to saturation), 300 mg of sodium bicarbonate, and approximately 1 g of microcrystalline cellulose. The potatoes and the mixture were left in contact for 10 minutes at room temperature (25°C) .

The pH value of the mixture of potatoes and soaking liquid was measured and found to be equal to 6.7.

The potatoes were rapidly washed in mains water, dried and fried.

Also in this C3. S θ 3. S control the same procedure was carried out on an identical specimen of potatoes treated, in the conditions described, only with microcrystalline cellulose.

Each test was repeated in triplicate.

Example 3 - Extraction of acrylamide present from the specimens of potatoes treated with asparaginase

Each specimen of fresh potatoes and pre-fried and frozen potatoes subjected to treatment with asparaginase according to the invention (Examples 1 and 2), after frying, was put into 200 ml of deionized water and left therein throughout the night so as to allow the acrylamide, which is very soluble in water, to pass into solution.

The solution thus obtained was mixed with CHCI3 (10 ml of H 2 0 : 40 ml of CHC1 3 ) . A liquid-liquid extraction with separator funnel was conducted according to the standard chemical procedure.

The underlying aqueous phase was recovered after extraction and was filtered with a 0.45-pm filter and analysed by means of RP-HPLC.

Example 4 - RP-HPLC analysis of the specimens obtained From extraction with chloroform an aqueous phase of approximately 50 ml was recovered, from which a specimen of 200 μΐ was taken, as described in Example 3. This was passed through a non-polar chromatographic column, immersed in a polar solvent. The non-polar compounds present in the specimen set up interactions with the non-polar groups present in the column and were partially withheld thereby. Hence, the chromatogram records the outputs of the various compounds present in the specimen on the basis of their times of exit from the column (retention time) . Polar compounds will present very short retention times, whereas non-polar compounds have longer retention times because they are withheld by the column. Foodstuff mixtures are very complex matrices, formed by multiple composites of various nature. Consequently, the peaks that appear in the chromatogram are not easy to interpret. Also the comparison of the chromatogram obtained with that of pure acrylamide is not simple since the retention time of a compound can be slightly varied by the matrix in which it is found. In this case, identification of the acrylamide peak was carried out using an internal standard, and this enabled identification of the peak of interest in each foodstuff matrix, as well as evaluation of possible differences resulting from the treatment.

Example 5 - Evaluation of the effect of temperature and cooking time on the formation of acrylamide

From an analysis of the data obtained in the various tests as regards the amount of residual acrylamide, albeit leaving unaltered the organoleptic properties of the product, i.e., without altering the taste of the product and its visual characteristics, the best conditions for frying were identified and were found to be a temperature of 150°C for 15-20 minutes.

Results

The percent reduction of the amount of acrylamide following upon treatment with the composition according to the invention was evaluated by comparison of the areas under the peaks obtained with RP-HPLC since the area under the peak is directly proportional to the corresponding amount of the compound in the specimen: % REDUCTION = 100 - [ (A with asparaginase /A contro i ) *100]

Table 1 - Fresh potatoes

Table 2 - Pre-fried and frozen potatoes

Appearing in Figure 1 are the chromatograms obtained for acrylamide abatement following upon treatment with asparaginase carried out on fresh potatoes, as described in Example 1 (B) , as compared to the control (A), i.e., with the same test repeated in the absence of asparaginase. As may be noted from the chromatogram B, the acrylamide peak is considerably reduced by treatment with the enzyme.

In (C) , from the superposition of the two chromatograms , there may be noted a substantial reduction of the peak obtained as a result of treatment with asparaginase enzyme according to the invention.

The reduction in acrylamide formation is approximately 70% in treated fresh potatoes. In the case of pre-fried potatoes, the reduction is, instead, approximately 60%. There is hence noted a greater reduction in acrylamide formation in fresh potatoes as compared to pre-fried potatoes. This demonstrates a greater effectiveness of the enzyme in the fresh product. This reduced abatement of acrylamide in pre- fried potatoes could be explained if we consider that during first frying, albeit brief, prior to freezing, acrylamide formation has already partially occurred. In the second frying process, treatment with asparaginase acts on the acrylamide formation deriving from the asparagine that has remained free after the first frying, and hence on a smaller amount.

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