A process and composition for preventing or reducing the formation of acrylamide in foods
The present invention relates to a process for preventing or reducing the formation of acrylamide and/or its precursors formed during cooking or food processing or food heating or food storage.
The present invention also relates to the use of this process for food, food ingredients or food products which are cooked or heated at relatively high temperature by heating, frying, deep-frying, baking, roasting, toasting or grilling.
The present invention also relates to the use of a flavonoid and/or a flavonoid blend for preventing or reducing the formation of acrylamide and/or its precursors formed during cooking or food processing or food heating or food storage.
The formation and presence of acrylamide in foods heated to relatively (over 100 °C) high temperature became a highly sensitive issue for the food indus- try last spring (Lingnert etal. 2002). According to the International Agency for Research on Cancer (1986), acrylamide may be carcinogenic for humans. Lingnert etal. (2002) have suggested a number of possible routes for acrylamide formation, the main precursor appearing to be acrolein, which is an aldehyde with acrylamide-like structure. Acrolein is an intermediate in many food processing reactions, including oxidation. Another likely precursor is asparagine. The use of antioxidants or other substances that will react with acrylamide or its precursors (amino acids, carbohydrates, lipids, proteins) might offer a solution, other than decreasing the temperature of cooking or food processing, to the presently unsolved problem of high acrylamide con- tent in heated foodstuffs. Flavonoids are a possible candidate for this purpose.
An object of the invention is to provide a solution to the problem of high acrylamide content in certain foodstuffs. For carrying out this purpose the process of the present invention is mainly characterised in that a flavonoid and/or a flavonoid blend is added to the food, the food ingredient(s) or the food product.
Another object of the invention is to provide a composition for preventing or reducing the formation of acrylamide and/or its precursors formed during cooking or food processing or food heating or food storage. For carrying out this purpose this composition is mainly characterised in that it contains a flavonoid and/or a flavonoid blend.
Further preferred embodiments of the present invention are as claimed in the dependent claims hereinafter.
Many flavonoids (phenolic compounds) are strong antioxidants capable of preventing oxidation reactions (Mursu 3 etal.f Change etal. 2001). Some flavonoids also seem to be protective against diseases such as cancer and inflammatory and cardiovascular diseases, primarily because of their antioxi- dant activity and ability to scavenge free radicals (Hosny, and Rosazza, 2002). The antioxidant activity of some catechins may be explained by their ability to trap peroxyl radicals by donation of hydrogen from phenolic rings A and B.
In general, the flavonoids are highly reactive during food processing (Es-Safi, N. etal. 2001) owing to the acidic character of their hydroxyl groups and the nucleophilic properties of the phenolic rings. Among the reactions are oxidation and addition, leading to various adducts and eventually to tannin-like polymeric compounds. Pierpoint (1990) has noted that flavonoids can react with chemical residues such as guanidine, amide, peptide, and carboxyl groups. Mallinckrodt Chemicals, in its Material Safety Data Sheet (MSDS,
2000), states that acrylamide can be stabilized with t-butylpyrocatechol and other antioxidants.
We set out to clarify the possibility of reducing or even preventing the forma- tion of acrylamide in heated foods by adding a flavonoid spice. The spice we added was the commercially available Flavomare® (from SLK Companies- SLK Foundation), which is a combination of many flavonoids. Flavomare® is currently used as a spice and salt substitute but can also be used in sweet drinks. In our earlier studies, Flavomare® was shown to be an exceptionally strong antioxidant both in vitro and in vivo. In cholesterol studies, Flavomare® has reduced cholesterol values (Mursu J etal., Change etal., 2001 and Vuorela etal., 2001 and M. Ahotupa, 2000). Flavomare® seasoning is available in liquid or powder form. The liquid form is used in juices, while the powder form is used as a table spice or salt substitute. Flavomare® (SLK Companies, Helsinki, Finland) is protected worldwide with patents and patent applications.
The food we chose for study was potato chips, which have been found to contain one of the highest levels of acrylamide.
Material and methods
The study was carried out at the Department of Food Technology, University of Helsinki. The potatoes (Rosamunda, Finland) were peeled and sliced to a thickness of about 1 mm. The slices were washed twice by hand under running water and dried between two adsorbent papers. The potato slices in the test group (group C) were IMMERSED for 15 seconds in a flavonoid solution containing 1.5% Flavomare® JB extract (Flavomare® JB is a commercially available product containing about 45% of T base™ 60 (green tea), 45% of apple concentrate and 10% of onion concentrate. Flavomare® JB is a chlorophyll-free liquid and its recommended dosage in the final product is between 0.05 and 0.15%). On the basis of an earlier study, 15 seconds was consid-
ered long enough for the flavonoids to diffuse into the potato slices in concentration great enough to hinder or prevent the formation of acrylamide (a longer time could have been chosen, but a longer time might be less economical). The slices were then oven-dried at 100 °C. The drying time for the flavonoid test group was 5.5 minutes, and the time for the samples without flavonoids (control group) was 3.5 minutes.
After drying, both groups of potato slices were fried in palm oil, as 50-60 g batches, in a small-scale deep fat fryer for household use. Each batch was fried separately, in fresh oil, for about 3 minutes until the slices were slightly brown in appearance. Flavonoid solution (0.3%) was added to the oil used for frying the flavonoid-treated potato slices but not to the oil used for frying the control slices. After the potato slices (chips) were removed from the oil, flavonoid powder was sprinkled on the flavonoid-treated slices. The dose was 1% calculated by weight. No flavonoid powder was added to the control group.
The potato chips produced in the two treatments were placed in plastic bowls, covered with aluminum foil and sent for analysis. Samples for acryla- mide analysis were sent to the National Veterinary and Food Research Institute (EELA) in Helsinki and samples for flavonoid analysis to the Department of Applied Chemistry and Microbiology, University of Helsinki. The samples were analyzed immediately.
The remaining bowls, of both groups, were placed in an incubator at 40 °C for 4 days, in darkness. Storage carried out in this way was intended to speed up the aging process.
After 4 days, samples representing the test group and the control group (one bowl each), as well as samples of the oil that was used (three samples of each oil) were sent to AnalyCen Laboratory in Lidkδping, Sweden, for determination of acrylamide content. Samples of the incubated potato chips were
also sent to the Department of Applied Chemistry and Microbiology for flavonoid analysis.
Moisture content and pH values were determined for uncooked potato sam- pies as well as the fried samples. The moisture content was evaluated after the samples had been in an incubator at 105 °C overnight. The pH measurement was made with a Radiometer PHM 92 Lab (electrode type GK 2401 C).
Results
Table 1 shows the acrylamide contents of the potato chips samples analysed immediately after frying. The content of acrylamide was 1282 μg/kg in the treated group and 3186 μg/kg in the control group. In other words, the acrylamide content in the control chips is 150% higher than in the chips treated with Flavomare®.
Table 1. Acrylamide Content in Fresh Potato Chips1
1 The analysis was performed 9.9.2002 at the National Veterinary and Food Research Insti- tute (EELA), Department of Chemistry, Helsinki, Finland. Method: the acrylamide was analysed by liquid chromatography tandem mass spectrometry.
Table 2 shows the acrylamide contents of the potato chip samples after incubating for four days in 40 °C, in darkness. After incubation, the content of acrylamide was 2100 μg/kg in the control group and 1100 μg/kg in the test group. Acrylamide content is thus 90% higher in the control potato chips than in the chips containing Flavomare®. NOTE THE PERCENTAGE.
Table 2. Acrylamide of Potato chips after 4 days oxidation at 40 °oC 2
2 The analysis was performed at AnalyCen, Lidkδping, Sweden. The test was started 18.9.2002 and finished 20.9.2002. Method: LC-MS-MS.
The flavonoid results, presented in table 3, clearly show that the flavonoid spice effectively diffuses in to potato chips. The amount of flavonoids, 7.1 μg/g of Flavonols and 1092 μg/g of catechins (Flavanols), appears to be sufficient to reduce the formation of acrylamide in the preparation of potato chips.
Moisture and pH values are shown in tables 4 and 5. The moisture content before frying was 70 - 72%.
The low pH value (3.42) of the flavonoid blend in liquid form is due to the hydroxyl groups in the phenolic compounds (Es-Safi etal. 2001). (THIS
VALUE IS NOT GIVEN IN THE TABLE) The difference in the pH values of samples before and after frying is small but smallest when the flavonoids are present (table 5).
Table 4. Moisture of Potato Chips4
5 The analysis was performed at the University of Helsinki, Department of Food Technology.
The scope of the present invention is illustrated by the following examples, which describes how Flavomare® can be used in food against acrylamide. These examples should by no means be construed as limiting the scope of the present invention.
Example 1. Bakery
a) Adding Flavomare® JB (0.15 %) to the dough. If the acrylamide is formed at the surface then this a questionable step.
b) Adding Flavomare® JB (1.5 %) by spraying or rubbing to the surface of the dough before baking.
c) Adding Flavomare® JB (1.5 %) by spraying to the surface of the dough during baking.
Example 2. French fries
a) Immersion in a solution of Flavomare® JB (1.5%) for 15 s, before frying.
Example 3. "Hapankorppu" or Crisp Bread
a) Adding Flavomare® JB by spraying or rubbing to the surface of the dough before baking. b) Adding Flavomare® JB (0.15 %) to the dough before the fermentation.
Note: The addition of Flavomare® in other technological steps must be known after an individual evaluation in each case.
The following examples 4 - 10 are examples of foods prepared with different flavonoid containing Flavomare® products. The method(s) of adding the flavonoid and/or the flavonoid blend can for example be chosen from the methods described in the examples 1-3. All these examples (1-10) should by no means be construed as limiting the scope of the present invention.
Example 4. British sausage with Flavomare® 06.06.00 Seasoning g/kg
Flavomare® * 11.000
Carfosell 2( Rhodia) 4.450
Ground white pepper 2.700
Ground nutmeg 1.800
Bouillon flavour FIS FB918101/k 1.500
Ground mace 0.900
Garlic powder 0.900
Fermented rice 0.400
MSG 0.400
Ascorbic Acid 0.400
Ribotide 0.050
Rosemary extract 0.050
Sodium Meta Bisulphite ( E 224 ) 0.050
24.600
The above seasoning is added to the meat ingredients of the sausage in the order as described below.
Example 5. British sausages recipe 11/01/00
With Flavomare® (1.4 %)
Addition order Formulation %
1 Lean Pork ( < 10 % fat) 29.68
1 Belly Pork ( 50 % fat ) 9.00
2 Cooked Pork rinds 5.00 6 Pork Fat 20.00
3 Bread crumb rusk 12.50
4 Ice 20.00 1 Salt 1.00 1 NaN02 ( 10 % Solution ) 0.12
5 Seasoning 2.70
100.00
Sheep casing, link
Storage at 2 - 4 °C/24 h then cook ( grill)
Note: 20 g Sausage Phosphate/ 100 kg mass at the beginning
The Flavomare® used in the above described British sausage is Flavomare® RM80.
Example 6. British Back Bacon290300
British Back Bacon290300 15 % Injection Seasoning blend
Ingredients g/kg Brine
Salt 3.00
Flavomare® JB 0.30
Fried Bacon XK1549 1.50
Hickory smoke yeast 2.00
Potassium Chloride 1.20
Ascorbic Acid 2.00
Potassium Sorbate 2.00
Potassium Nitrate 0.75
12.75
The above seasoning is prepared by mixing the ingredients including Flavomare® JB together and it is thereafter injected into the bacon before cooking of the bacon. The composition of Flavomare® JB is described in the table below.
Example 7. Breads (Flavomare
® B is used for bread)
White standard bread for sandwich Gunstone breads
Formula
Ingredients Flavomare® bread
Wheat flour 567.40
Salt 4.95
Improver Ascorbic Acid 11.30
Margarine 24.10
Yeast 20.00
Wheat protein 17.00
Emulsifier * 12.80
Water 335.80
Flavomare® B (bread) 7.90
1001.25
The above ingredients including Flavomare® B, the composition thereof described in the table below, are mixed together. Thereafter the dough is baked.
Example 7. "Ruisleipa" or rye-bread
Rye-bread
Ingredients %
Wholegrain rye 78.4
Water 39.1
Salt 0.5
Flavomare® B 1
The above ingredients including Flavomare® B are mixed together. Thereaf- ter the dough is baked.
Example 8. White bread
White bred . A )
Ingredients %
Water 33.80
Wheat flour 57.20
Sieved rye flour 1.80
Flax seed 1.30
Yeast 2.00
Rapeseed oil 2.40
Salt 0.50
Flavomare® B 1.00
100.00
The above ingredients including Flavomare® B are mixed together. Thereafter the dough is placed in the oven for baking.
Example 9. VITAL Juice for Marli
VITAL Juice for Marli
Apple Vital juice + Flavomare® JB
Ingredients
Product water 30.00
Ascorbic acid solution ( 20 % ) 0.86
Apple concentrate 70 Brix 160.00
Calcium lactate ( 8.90
Citric acid solution ( 50 % ) 1.50
Flavomare® JB ( v/v ) 0.10
Product water 10.00
Product water 834.00
The above ingredients including Flavomare® JB are simply mixed together and the juice product is thereafter ready for drinking. The composition of Flavomare® JB is described below.
Example 10. Potato chips seasoning with Flavomare® RM 80
Potato chips seasoning with Flavomare® RM 80
Ingredients g/kg ready product
Salt 10.00
Flavomare® RM 80 9.40
Sugar 2.10
Hickory smoke yeast (BBA) 3.00
Cayenne pepper, ground 0.30
White pepper 0.30
Hydrolysed vegetable protein 2.50
Yeast extract 3.00
Paprika oleoresin ( 100 000) 0.35
Natural caramel colour ( E 150 a) 0.30
Carotene (E 160 c) 0.15
Maltodextrin ( DE:4-10) 3.60
Silicon dioxide ( E 551) 10.00
45.00
The above potato chip seasoning containing Flavomare® RM 80 is made by mixing the ingredients together. Thereafter the seasoning is added to the potato chip dough or the uncooked chips before the frying step.
In the following the Flavomare® products used in the non-limiting examples of this invention are described:
Flavomare® RM 80: This is a discoloured Flavomare® that could be used in ready meal, snacks, meat products, etc and in general where the turbidity is not important. The recommended dosage in the final product is between 0.50 to 1.50 %.
Flavomare® B 80: This is a discoloured Flavomare® with low onion profile to avoid the strong onion (vegetable feelings) flavour and the metallic taste that
are undesirable in some products. It could be used in ready meal, snacks, meat products, etc, and in general where the turbidity is not important. . This Flavomare® is good for baking. The recommended dosage in the final product is between 0.50 and 1.50 %.
Flavomare® JB: This is a discoloured Flavomare® in liquid form, with low onion profile, suitable for all kinds of foodstuff especially where the turbidity is important as beverages, juices, sauces, marinades, gravies, confectionery, dairy products in general as well as for products that are injected with brine as cook hams, bacons, etc. The recommended dosage in the final product is between 0.05 and 0.15 %.
As mentioned before the process of the invention is useful for foodstuffs wherein acrylamide is formed in considerable amounts during cooking or heating at relatively high temperature. By relatively high temperature is preferably meant a temperature above 100 °C. Typical food processes where the temperature is high enough for acrylamide to be formed in considerable amounts are heating, frying, deep-frying, baking, roasting, toasting or grilling.
The list of foodstuffs with measurable or considerably high levels of acrylamide is expanding as more tests are being performed. At this point this list include potato chips, French fries, cookies, biscuits, crackers, coffee (powder), breakfast cereals, roasted asparagus, banana chips, roasted English muffins, taco shells, corn chips, pretzels, corns, popcorns and breads.
When it comes to the group of breads, toasts have had the highest measured levels of acrylamide. It is considered that the acrylamide arises from within the dough to the surface of the dough during baking in the oven. Therefore it is very preferable to add the flavonoid and/or flavonoid blend to the dough before the baking step. Alternatively or complementarily the fla-
vonoid and/or flavonoid blend can also be added to the surface of the dough before and/or during baking.
Food products that have been found to contain less than 30 μg/kg of acryla- mide are the following: pizza, pancakes, waffles, scrambled egg, raw, boiled or mashed potatoes, pasta, wheat and rye flour, rice, oat flakes, vegetarian schnitzels, cauliflower gratin, dried fruits, beer (alcohol free), meat and fish products. The amount of acrylamide in these products may rise as more analyses are preformed on them, because deviations might occur in the samples.
The flavonoids according to the process of the invention are advantageously selected from the flavanols and/or the flavonols. Other subgroups of the flavonoids may also be selected for the process of the invention. Examples of flavonols are for example kaempherols, quercetins and myricetins and their derivatives. Examples of flavanols are for example epigallocatechins, epigal- locatechin gallates, (-)-catechins and epicatechin gallates and their derivatives.
Discussion
Both immediately after frying and after incubation, the amount of acrylamide is very much less in the samples treated with flavonoids. The difference could be due to the antioxidant properties of the flavonoids and their reac- tions in the food environment with compounds involved in the formation of acrylamide. These reactions are still poorly known and the mechanisms we suggest below should be considered as plausible hypotheses.
In an earlier study made at the University of Helsinki (Hopia, A. & Vuorela, 1997) it was found that concentrations of 125 ppm and 250 ppm of the same flavonoids used in this study were sufficient to inhibit the oxidation of methyl linoleate in potato chips during storage. The content of flavonoids in the
treated samples of this study is equivalent to the lower amount used by Hopia, A. & Vuorela (1997). This amount was enough for inhibiting the lipid oxidation and to some extent reducing the formation of acrylamide, which is produced above 100 °C. The flavonoids reduce the amount of metabolites that later on in the frying step are capable of participating in the acrylamide formation.
The flavonoids might be oxidized by action of the polyphenoloxidase that is liberated during the slicing step. Furthermore, hydroquinone, which is formed in the polymerisation of the flavonoids during the enzymatic browning reaction in the hot oil, may react with acrylamide (MSDS, 2000).
The flavonoids also may undergo reactions with a reducing sugar, with reaction occurring between the OH groups in the flavonoids and the OH reducing groups in the sugar (Bruice, P.Y. 2001). Flavonoids may react with amino acids in deamination and acylation reactions (Bruice, P.Y. 2001). These reactions would decrease the amounts of reducing sugars and amino acids and their potential for participating in the Maillard reaction. In our tests we found that the control sample was browner in appearance than the treated sample after frying. The amount of the free amino acid asparagine, which recently has been reported to be a key precursor in acrylamide formation (Health Canada, 2002), could be decreased as a consequence of the reactions between flavonoids and amino acids.
During the frying step the flavonoids might prevent the oxidation of glycerol to acrolein. NOTE NEXT SENTENCE. It is well known that the flavonoids and their metabolites inhibit lipid oxidation (Hopia, A. & Vuorela, 1997 and Hosny, M., Rosazza J. P. N. 2002, Connie and Christensen, 1996 and Biochemistry & Molecular Biology, 2001). The flavonoids could react in several ways with acrolein as an aldehyde (Es-Safi, N. et.al. 2001); possible reactions are nucleophilic addition to the double bond, C-C reaction, and acylation reaction (Bruice, P.Y. 2001). The flavonoids could suppress the forma-
tion of acrylamide by reacting with acrolein before the moisture content drops too low. At higher moisture levels, flavonoids could prevent the formation of acrylamide from precursors formed in Strecker degradation, which occurs in the Maillard reaction. The amount of acrylamide could also be de- creased by similar reactions, wherein flavonoids react with the amide group of acrylamide in a so called annulation-reaction (Bruice, P.Y. 2001).
The results showed some level of decrease in the level of acrylamide during the storage for 4 days at 40 °C. The decrease might be only apparent, re- fleeting the two different methods used in the laboratories in Finland and Sweden. Another possible explanation is variation in the amount of acrylamide in samples from the same batch (Lingnert etal. 2002). The main weight in evaluating the results should thus be put on the difference in values between the treated and control groups.
The effect of the flavonoids in reducing the amount of acrylamide after it is formed would seem to be less important than the effect in preventing its formation in the first place. As can be seen in table 4, the moisture level in the potatoes was greater than 70% before frying. Reactions between the flavonoids and acrylamide and its precursors can occur until the moisture level falls below 5% (Tareke, E. et.al.2002 and Lingnert etal. 2002).
Reactions with flavanoids are particularly sensitive to pH. In any reactions with flavonoids, it is important to be aware of the pH. The low pH value (3.42) of the flavonoid mixture in this study is due to the acid hydroxyl groups (Es-Safi, N. etal. 2001). As a result of the treatment with Flavomare®, the pH of the flavonoid-treated slices before frying is slightly lower than the pH of the corresponding control slices. The lower pH may help to inhibit the Maillard reaction (Lingnert etal. 2002) during the frying step. The pH (5.43) of asparagic acid (Bruice, P. Y., 2001) is slightly lower than the pH of the flavonoid-treated slices before oven drying and this low pH could allow reaction of asparagic acid with the flavonoids. Although the difference in the
pH values between the samples is small, it may be great enough to promote the reaction with asparagine and prevent its further reaction to acrylamide.
Summary
The newly demonstrated presence of acrylamide in heated foodstuff is a problem for the food industry. Acrylamide is a possible human carcinogen. Two main precursors for acrylamide have been proposed: acrolein (aldehyde) and asparagine (amino acid). The addition of antioxidants or other substances that will react with the acrylamide or its sources (amino acids, carbohydrates, lipids or proteins) may offer a way to reduce the amount of acrylamide in prepared food. In this study the amount in potato chips was dramatically reduced when a flavonoid spice, Flavomare®, was applied to the raw potatoes.
References
- Bruice, P. Y. (2001). Organic Chemistry, 3rd Edition, Pearson Education, pp 111, 450, 481, 690, 707, 878 and 1033.
- Connie R. and Christensen, G. 1996. Enzymatic browning inhibitors: factors which interfere with or prevent polyphenoloxidase from oxidizing phenolic compounds in tissues and creating brownish polymers. June 3, Food Resource Nutrition and Food Management, Oregon State University, Corvallis, OR. USA.
- Elliot, W.H. and Elliot D.C. (2001). Biochemistry and Molecular Biology. 2nd Edition, Oxford University Press, p 282
- Hopia, A. & Vuorela, H. Applications of a flavonoid containing seasoning, Flavomare®, for the food industry. 1997. Personal communication. Department of Applied Chemistry and Microbiology and Department of Pharmacy at the University of Helsinki
- Kahkόnen, M.P., Hopia, A.L. & Heinonen, M. 2001. Berry phenolics and their antioxidant activity. J. Agric. Food, Chem. 49: 4076-4082
- International Agency for Research on Cancer. Sixth Annual Report on Carcinogens. Acrylamide CAS No. 79-06-1, (1986). IARC V.39.
- Lingnert, H., Grivas, S., Jagerstd, M., Skog, K., Tδrnqvist, M. & Aman, P. Acrylamide in food. Mechanism of formation and influencing factors during heating of foods. A report from the Swedish Scientific Expert Committee. The Swedish Institute for Food and Biotechnology. June 30, 2002.
- Mallinckrodr Chemicals. 2000. MSDS (A1550), Acrylamide. Mallinckrodt Chemicals Inc. 222 Red School Lane, Phillipsburg, NJ 08865, USA.
- Mursu, J., Voutilainen, S., Salonen J.T. 2001. The effect of flavonoids flavonoid supplementation on serum lipids and their oxidation - preliminary results. Personal communication. University of Kuopio. May.
- Nardini, M., Cirillo, E., Natella, F. and Scaccini, C. Absorption of Phenolic Acids in Humans after Coffee Consumption. J. Agric. Food Chem. 2002. 50 (20), 5735 -5741.
- Pierpoint, W.S. Flavonoids in human food and animal feedstuffs: amounts and consequences. 3rd International Symposium Flavonoids in Biology & Medicine.
- Rosen, J. and Hellenas, K. E. (2002). Analysis of Acrylamide in cooked food by liquid chromatography tandem mass spectrometry. The Analyst, vol. 217
(880-882).
- Tareke,E., Rydberg, P., Karlsson, P., Eriksson, S. and Tornqvist, M. Analysis of Acrylamide, a Carcinogen Formed in Heated Foodstuffs. 2002. J. Agric. Food Chem., 50 (17), 4998 -5006.