U.S. patent application number 14/619149 was filed with the patent office on 2015-08-13 for method for the control of the formation of acrylamide in a foodstuff.
The applicant listed for this patent is DUPONT NUTRITION BIOSCIENCES APS. Invention is credited to Dana L. Boll, Charlotte Horsmans Poulsen, Lars Wexoe Petersen, Thomas Rand, Jorn Borch Soe.
Application Number | 20150223499 14/619149 |
Document ID | / |
Family ID | 34923475 |
Filed Date | 2015-08-13 |
United States Patent
Application |
20150223499 |
Kind Code |
A1 |
Soe; Jorn Borch ; et
al. |
August 13, 2015 |
METHOD FOR THE CONTROL OF THE FORMATION OF ACRYLAMIDE IN A
FOODSTUFF
Abstract
There is provided a process for the prevention and/or reduction
of acrylamide formation and/or acrylamide precursor formation in a
foodstuff containing (i) a protein, a peptide or an amino acid and
(ii) a reducing sugar, the process comprising contacting the
foodstuff with an enzyme capable of oxidising a reducing group of
the sugar.
Inventors: |
Soe; Jorn Borch; (Tilst,
DK) ; Petersen; Lars Wexoe; (Muskego, WI) ;
Horsmans Poulsen; Charlotte; (Brabrand, DK) ; Rand;
Thomas; (Copenhagen, DK) ; Boll; Dana L.;
(Olathe, KS) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DUPONT NUTRITION BIOSCIENCES APS |
Copenhagen |
|
DK |
|
|
Family ID: |
34923475 |
Appl. No.: |
14/619149 |
Filed: |
February 11, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13433470 |
Mar 29, 2012 |
8956670 |
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14619149 |
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11048230 |
Feb 1, 2005 |
8163317 |
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13433470 |
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10001136 |
Nov 15, 2001 |
6872412 |
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11048230 |
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PCT/IB2003/005278 |
Oct 24, 2003 |
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11048230 |
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60256902 |
Dec 19, 2000 |
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60438852 |
Jan 9, 2003 |
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Current U.S.
Class: |
426/10 ; 426/102;
426/637 |
Current CPC
Class: |
A23L 19/18 20160801;
A21D 8/042 20130101; A23V 2002/00 20130101; A23C 19/063 20130101;
A23C 19/0912 20130101; C12Y 111/01006 20130101; A23V 2002/00
20130101; C12Y 101/03009 20130101; A23V 2002/00 20130101; A23V
2250/612 20130101; A23V 2200/042 20130101; A23V 2200/048 20130101;
A23V 2200/042 20130101; A23V 2250/608 20130101; A23V 2200/048
20130101; A23L 5/25 20160801; A23V 2250/608 20130101; A23L 5/40
20160801; C12Y 101/03004 20130101; C12Y 101/03005 20130101; A23B
7/155 20130101; A23C 19/061 20130101; A23V 2250/612 20130101; A23V
2002/00 20130101; A23V 2002/00 20130101 |
International
Class: |
A23L 1/217 20060101
A23L001/217; A23L 1/015 20060101 A23L001/015; A23L 1/00 20060101
A23L001/00; A23L 1/2165 20060101 A23L001/2165 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 30, 2002 |
GB |
0225236.9 |
Claims
1. A potato or potato part thereof wherein the formation of
acrylamide and/or an acrylamide precursor is prevented or reduced
during heating which is obtained by a process which comprises
contacting said potato or potato part thereof with an enzyme prior
to heating, wherein the improvement comprises the enzyme being
hexose oxidate (EC 1.1.3.5).
2. The potato or potato part thereof according to claim 1, which is
French fries, potato chips, potato crisps, coated French fries,
coated potato chips, potato flour or potato starch.
3. The coated potato chips according to claim 2, wherein the coated
potato chips are coated with corn starch.
4. The coated French fries according to claim 2, wherein the coated
French fries are coated with corn starch.
5. The potato or potato part thereof according to claim 1, wherein
the heating comprises frying.
6. The potato or potato part thereof according to claim 1, wherein
the enzyme is contacted with the enzyme hexose oxidate (EC 1.1.3.5)
by spraying a solution or dispersion comprising said hexose oxidate
(EC 1.1.3.5) onto said potato or potato part thereof.
7. The potato or potato part thereof according to claim 6, wherein
the amount of hexose oxidase in the solution or dispersion is from
1-50 unites of hexose oxidate/ml.
8. The potato or potato part thereof according to claim 1, wherein
the process further comprises contacting said potato or potato part
with a catalase.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/433,470 filed Mar. 29, 2012, which is a continuation of U.S.
application Ser. No. 11/048,230 filed Feb. 1, 2005, which claims
priority from U.S. application Ser. No. 10/001,136 filed Nov. 15,
2001, which claims priority from U.S. Provisional Application
60/256,902 filed Dec. 19, 2000 and United Kingdom Application
0028119.6 filed Nov. 17, 2000. U.S. application Ser. No. 11/048,230
filed Feb. 1, 2005 is also a continuation-in-part of International
Patent Application No. PCT/IB2003/005278 filed Oct. 24, 2003 and
published as WO 2004/039174 on May 13, 2004, which claims priority
from United Kingdom Application 0225236.9 filed Oct. 30, 2002 and
U.S. Provisional Application No. 60/438,852 filed Jan. 9, 2003. All
of the above-mentioned applications, as well as all documents cited
herein and documents referenced or cited in documents cited herein,
are hereby incorporated herein in their entirety by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to the control of the
formation of acrylamide in a foodstuff.
##STR00001##
BACKGROUND OF THE INVENTION
[0003] Acrylamide and polyacrylamide are used in industry for the
production of plastics. It has been supposed that the main exposure
for acrylamide in the general population has been through drinking
water and tobacco smoking. Exposure via drinking water is small and
the EU has determined maximum levels of 0.1 microgram per litre
water.
[0004] Acrylamide is water soluble and is quickly absorbed in the
digestive tract. Excretion via the urine is fast and half of
acrylamide is cleared from the body in a few hours.
[0005] The toxicological effects of acrylamide are well known. It
causes DNA damage and at high doses neurological and reproductive
effects have been observed. Glycidamide, a metabolite of
acrylamide, binds to DNA and can cause genetic damage. Prolonged
exposure has induced tumours in rats, but cancer in man has not
been convincingly shown. The International Agency for Research on
Cancer (IARC) has classified acrylamide as a "probably carcinogenic
to humans" (Group 2A).
[0006] Acrylamide has been shown to induce gene mutations in
cultured animal cells and also in animals treated in vivo. Thus it
is assumed that exposure also to very low doses of acrylamide
increases the risk for mutation and cancer.
[0007] High doses of acrylamide have been applied in the
toxicological studies, which is an accepted practice. 25-50 mg per
kg body weight is the lowest dose that has been shown to increase
the mutation frequency in mouse. Recent studies in the laboratory
of the Swedish Food Administration have shown that chromosome
aberrations are induced in mice at 10-20 times lower doses.
[0008] Among the acrylamide metabolites glycidamide is considered
the most likely candidate for causing genetic damage. Glycidamide
has been found in mice and rats, and also in humans exposed to
acrylamide.
[0009] Neurological damage was observed when rats were given
acrylamide in their drinking water. The lowest effective dose was 2
mg/kg body weight and day, and the highest no-effect dose was 0.5
mg/kg body weight and day. Also humans exposed to high doses of
acrylamide have shown neurological damage, e.g. some workers
occupied in the building of the tunnel at Hallandsasen. It is
difficult to assess the highest acrylamide dose in humans that does
not cause neurological effects (NOEL). The level is probably
several times higher than the average acrylamide intake from
food.
[0010] Decreased fertility was observed in rats exposed to 5-10 mg
acrylamide/kg body weight and day.
[0011] Epidemiological studies in man have not shown a correlation
between exposure to acrylamide and increased cancer rate. These
studies have been criticised because the number of studied persons
was too low considering the expected effect.
[0012] Two long-term studies in rats have shown a substantial
increase of tumours in different organs when the animals were
exposed to acrylamide in drinking water. Similar studies have been
made in mice. The lowest effective dose was 2 mg/kg body weight per
day.
[0013] In the studies with rats the increase of tumours was most
evident in specific organs, e.g. mammary gland, uterus, adrenal
gland, scrotal mesothelium. In mice there was an increase of lung
and skin tumours. These cancer studies have been used for the
assessment of the risk of cancer in humans due to acrylamide
exposure.
[0014] It should be noted that the genotoxic studies have indicated
that there is no threshold value for the risk of cancer induced by
acrylamide, i.e. there is no dose of acrylamide so low that it does
not increase the risk of cancer. In making these assessments it is
assumed that man and rat have the same sensitivity for cancer
induction by acrylamide.
[0015] The results of the risk assessments are somewhat different
since they are based on different mathematical models. By
consumption of 1 microgram acrylamide/kg body weight per day the
lifetime risk for cancer has been calculated to [0016] 4.5 per 1000
(U.S. EPA) [0017] 0.7 per 1000 (WHO) [0018] 10 per 1000 (Granath et
al. 1999, Stockholm University)
[0019] Recent analyses have now indicated that the exposure to
acrylamide is probably considerably higher (for non-smokers) from
consumption of certain foods that have been heated. As reported in
J Agric Food Chem. 2002 Aug. 14; 50(17):4998-5006 a group at the
University of Stockholm, headed by Prof. Margareta Tornqvist, has
found that acrylamide is formed during heating of starch-rich foods
to high temperatures.
[0020] The Swedish National Food Administration has developed a
LC/MS/MS-method for the analysis of acrylamide in foods. Analysis
has shown that acrylamide is present in a large number of foods,
including many regarded as staple foods. The levels of acrylamide
differ widely within each food group analysed.
[0021] Using information on the levels in different foods and
Swedish food consumption data, it is suggested that a significant
number of annual cancer cases can be attributed to acrylamide.
[0022] When foodstuffs were analysed at the Swedish National Food
Administration (NFA) in Uppsala and at AnalyCen AB in Lidkoping it
was found that some foodstuffs, which had been heated, could
contain relatively high levels of the substance acrylamide. In
total, more than 100 food samples have been analysed at the NFA.
The food survey comprised bread, pasta, rice, fish, sausages, meat
(beef and pork), biscuits, cookies, breakfast cereals and beer, etc
as well as some ready-made dishes such as pizza and products based
on potatoes, maize and flour.
[0023] The levels of acrylamide vary considerably between single
foodstuffs within food groups, but potato crisps and French fries
generally contained high levels compared to many other food groups.
The average content in potato crisps is approximately 1000
microgram/kg and in French fries approximately 500 microgram/kg.
Other food groups which may contain low as well as high levels of
acrylamide are crisp bread, breakfast cereals, fried potato
products, biscuits, cookies and snacks, e.g. popcorn.
[0024] Foodstuffs which are not fried, deep fried or oven-baked
during production or preparation are not considered to contain any
appreciable levels of acrylamide. No levels could be detected in
any of the raw foodstuffs or foods cooked by boiling investigated
so far (potato, rice, pasta, flour and bacon).
[0025] According to the NFA food survey "Riksmaten 1997-98", which
is based on approximately 1200 individuals between the age of 17 to
70 who recorded their food consumption during one week, an average
intake of acrylamide of approximately 25 microgram per day (maximum
intake is approximately six times higher) is obtained, based on the
food groups shown below. The remaining food groups are estimated to
account for approximately 10-15 microgram of acrylamide; in total
an average intake of 35-40 microgram. The percentage contribution
based on an intake of 40 microgram acrylamide per day results in:
[0026] potato products: 36% (French fries 16%, fried potatoes 10%,
crisps 10%) [0027] bread: 16% [0028] biscuits, cookies and wafers:
5% [0029] breakfast cereals: 3% [0030] remaining foodstuffs groups,
basically not investigated yet: 40%
[0031] Young adults (17 to 34 years of age) have, according to
"Riksmaten", a higher consumption of snacks (nuts, chips and
popcorn) than other adults. For children under 17 years of age
newer data are lacking. In the food survey "Ungdom mot ar
2000"(Samuelson et al 1996), which was carried out 1993-94 among
15-year olds in Uppsala and Trollhattan, the consumption of snacks
was comparable to that of young adults in Riksmaten. Children have
a lower average body weight than the 70 kg generally assumed when
carrying out risk assessments. This implies that the food intake
per kg body weight and the exposure to various substances could be
even larger for those groups of individuals compared to adults.
According to Riksmaten, 10 per cent of the adult population
consumes 90 per cent of the snacks consumed in Sweden.
[0032] An alternative way of estimating the intake of acrylamide is
by adduct measurement, that is to measure a reaction product of
acrylamide with the protein of the blood, the haemoglobin
(Tornqvist et al 1997). This reaction product seems to occur in all
investigated humans at approximately the same levels and is
furthermore a measurement of the continuously administered dose of
acrylamide. The reason is unknown in this case, but workers who
were exposed to acrylamide at the tunnel accident at Hallandsasen
in Sweden had higher levels of this reaction product in their
blood.
[0033] In the general population, although not in smokers (who have
a level of this adduct 2-3 times the background level), the
background level has been estimated to account for a daily intake
corresponding to approximately 100 microgram per day.
[0034] Other sources than foodstuffs (estimated average intake of
35-40 .mu.g/day), e.g. cosmetics, drinking water, and a possible
endogenous formation in the body of acrylamide, could, to a lower
extent contribute to the background level. Estimated administered
amount of acrylamide for the formation of the background level
together with levels of acrylamide in foodstuffs are, however,
presently extremely uncertain.
[0035] A Report from Swedish Scientific Expert Committee entitled
"Acrylamide In Food--Mechanisms of formation and influencing
factors during heating of foods" discloses possible mechanisms for
the formation of acrylamide in food. Relevant extracts from this
report are given below in Appendix 1.
[0036] According to Health Canada, model experiments carried out in
the Food Directorate showed that when asparagine is heated with
glucose, acrylamide is produced. In an open letter, Health Canada
stated "The production of acrylamide in these studies was
temperature dependent and gave comparable results to those found
when potato slices were similarly heated. At this time, not much is
known about other possible pathways of formation of acrylamide in
foods."
[0037] Further discussion of reactions occurring during heating of
food is given in Principles of Food Chemistry pages 100-109. This
discussion is provided in Appendix 2.
[0038] The present invention alleviates the problems of the prior
art.
[0039] Some aspects of the invention are defined in the appended
claims.
BRIEF SUMMARY OF THE INVENTION
[0040] In one aspect the present invention provides a process for
the prevention and/or reduction of acrylamide formation and/or
acrylamide precursor formation in a foodstuff containing (i) a
protein, a peptide or an amino acid and (ii) a reducing sugar, the
process comprising contacting the foodstuff with an enzyme capable
of oxidising a reducing group of the sugar.
[0041] In one aspect the present invention provides use of an
enzyme for the prevention and/or reduction of acrylamide formation
and/or acrylamide precursor formation in a foodstuff containing (i)
a protein, a peptide or an amino acid and (ii) a reducing sugar,
wherein the enzyme is capable of oxidising a reducing group of the
sugar.
[0042] Acrylamide formation and/or acrylamide precursor formation
in cooked foodstuffs, in particular starch foodstuffs and
foodstuffs containing a protein/amino acid/peptide and reducing
sugar is described in Appendices 1 and 2, for example by the
Amadori reaction, and is known in the art. In such foodstuffs a
sugar such as glucose, galactose and/or maltose may react with an
amino acid such as asparagine, glutamic acid, lysine, or arginine.
Any primary amine capable of nucleophilic attack on the carbonyl
group of a reducing sugar may be involved This reaction may be an
important step in the formation of acrylamide.
[0043] The present invention prevents and/or reduces the
problematic condensation reactions between amino acids, in
particular the amino group thereof, and reducing sugars which
result in acrylamide or acrylamide precursor formation. These
reactions may comprise the Amadori reaction, Heynes rearrangements,
or reaction cascades resulting from the Maillard reaction. The
present invention may prevent and/or reduce the reaction which
directly results in acrylamide formation. It may also prevent
and/or reduce reaction(s) which provide materials which further
react to provide acrylamide, namely acrylamide precursors.
Acrylamide precursors are often provided by degradation of
carbohydrates. A typical acrylamide precursor is 2-propenal.
[0044] The problems of the formation of acrylamide and/or
acrylamide precursor formation in foodstuffs containing a protein
and a reducing sugar such as baked food products, in particular
formation caused either completely or in part by the Amadori
reaction, can be controlled by contacting the foodstuff with an
enzyme capable of oxidising the reducing group of the sugar. This
is a novel approach in which reducing sugar is oxidised to avoid
acrylamide formation and/or acrylamide precursor formation by
bringing the foodstuff into contact with an enzyme which is capable
of performing the necessary oxidation and thereby eliminating the
reducing sugar from the foodstuff by conversion.
[0045] In the present specification, by the term "prevention and/or
reduction of acrylamide formation" it is meant that the amount of
acrylamide produced is reduced and/or the period of time required
for formation of a given amount of acrylamide is increased.
[0046] In some aspects preferably the process prevents and/or
reduces Amadori reaction in a foodstuff.
[0047] Thus in one aspect the present invention provides a process
for the prevention and/or reduction of Amadori reaction in a
foodstuff containing (i) a protein, a peptide or an amino acid and
(ii) a reducing sugar, the process comprising contacting the
foodstuff with an enzyme capable of oxidising a reducing group of
the sugar.
[0048] In one further aspect the present invention provides use of
an enzyme for the prevention and/or reduction of Amadori reaction
in a foodstuff containing (i) a protein, a peptide or an amino acid
and (ii) a reducing sugar, wherein the enzyme is capable of
oxidising a reducing group of the sugar.
[0049] In the present specification, by the term "prevention and/or
reduction of Amadori reaction" it is meant that the extent of a
Amadori reaction is reduced and/or the period of time required for
completion of a Amadori reaction is increased.
[0050] In some aspects preferably the enzyme is capable of
oxidising the reducing group of a monosaccharide and the reducing
group of a disaccharide.
[0051] In some aspects preferably the enzyme is hexose oxidase
(EC1.1.3.5) or glucose oxidase (EC1.1.3.4). In a highly preferred
aspect the enzyme is hexose oxidase. Preferably the HOX is obtained
or prepared in accordance with WO 96/40935. Preferably the HOX is
DairyHOX.TM. available from Danisco A/S, Denmark.
[0052] In some aspects preferably the enzyme may oxidise
matlodextrins and/or celludextrins. In a preferred aspect the
enzyme is a carbohydrate oxidase which may oxidise matlodextrins
and/or celludextrins. Preferably the carbohydrate oxidase is
obtained or prepared in accordance with WO 99/31990.
[0053] Hexose oxidase (HOX) is a carbohydrate oxidase originally
obtained from the red alga Chondrus crispus. As discussed in WO
96/39851 HOX catalyses the reaction between oxygen and
carbohydrates such as glucose, galactose, lactose and maltose.
Compared with other oxidative enzymes such as glucose oxidase,
hexose oxidase not only catalyse the oxidation of monosaccharides
but also disaccharides are oxidised. (Biochemica et Biophysica Acta
309 (1973), 11-22).
[0054] The reaction of glucose with Hexose Oxidase is
D-glucose+H.sub.2O+O.sub.2.fwdarw..delta.-D-gluconolactone+H.sub.2O.sub.-
2
[0055] In an aqueous environment the gluconolactone is subsequently
hydrolysed to form gluconic acid.
##STR00002##
[0056] As shown, HOX oxidises the carbohydrate at the reducing end
at carbon 1 and thus eliminates the possible involvement of the
carbohydrate in acrylamide formation and/or acrylamide precursor
formation by Amadori rearrangement or later reaction with a
ketoseamine or aldoseamine to a diketoseamine or a diaminosugar
respectively.
[0057] In a preferred aspect of the present invention the enzyme is
capable of oxidising the sugar of the foodstuff at the 1 position.
This aspect is advantageous because it ensures that the reducing
sugar is oxidised such that the reducing part of the sugar is no
longer available to undergo a condensation reaction with an amino
acid such the Amadori reaction.
[0058] In some aspects preferably the reducing sugar is selected
from lactose, galactose, glucose, xylose, mannose, cellobiose and
maltose.
[0059] In some aspects the reducing sugar is lactose or
galactose.
[0060] In some aspects the reducing sugar is galactose.
[0061] In some aspects preferably the foodstuff is selected from
bakery goods including bread and cakes, pasta, rice, fish,
sausages, meat including beef and pork, biscuits, cookies, crisp
bread, cereals, pizza, beverages including coffee, and products
based on potatoes, maize and flour, including potato flour and
potato starch products.
[0062] In some aspects the foodstuff is a beverage.
[0063] In some aspects the foodstuff is a starch containing
foodstuff.
[0064] In some aspects the foodstuff is a cereal or part of a
cereal.
[0065] In some aspects preferably the foodstuff is selected from a
dairy foodstuff; milk based or milk containing foodstuff, such as
gratin; an egg based foodstuff; an egg containing foodstuff; bakery
foodstuffs including toasts, bread, cakes; and shallow or deep
fried foodstuff such as spring rolls.
[0066] When the foodstuff is a dairy foodstuff it may be cheese,
such as mozzarella cheese.
[0067] In some aspects preferably the foodstuff is a potato or a
part of a potato. Typical potato products in which the present
invention may be applied are French fries, potato chips (crisps),
coated French fries and coated potato chips, for example French
fries or potato chips coated with corn starch, and potato flour and
potato starch products.
[0068] The enzyme may be contacted with foodstuff during its
preparation or it may be contacted with the foodstuff after the
foodstuff has been prepared yet before the food stuff is subjected
to conditions which may result in the undesirable acrylamide
formation and/or acrylamide precursor formation. In the former
aspect the enzyme will be incorporated in the foodstuff. In the
later aspect the enzyme will be present on the surface of the
foodstuff. When present on the surface acrylamide formation and/or
acrylamide precursor formation is still prevented as it is the
surface of a material exposed to drying and atmospheric oxygen
which undergoes the predominant acrylamide formation and/or
acrylamide precursor formation.
[0069] When contacted with foodstuff during its preparation the
enzyme may be contacted at any suitable stage during its
production. In the aspect that the foodstuff is a dairy product it
may be contacted with the milk during acidification of the milk and
precipitation of the milk curd. In this process the enzyme (such as
HOX) is not active during the anaerobic conditions created during
the acidification and milk protein precipitation, but will be
active in the dairy product such as cheese when aerobic conditions
are created. Once in aerobic conditions the enzyme oxidise the
reducing sugar and reduce the tendency to acrylamide formation
and/or acrylamide precursor formation.
[0070] For application of the enzyme to the surface of the
foodstuff, one may apply the enzyme in any suitable manner.
[0071] Typically the enzyme is provided in a solution or dispersion
and sprayed on the foodstuff. The solution/dispersion may comprise
the enzyme in an amount of 1-50 units enzyme/ml, such as 1-50 units
Hexose Oxidase/ml.
[0072] The enzyme may also be added in dry or powder form. When in
wet or dry form the enzyme may be combined with other components
for contact with the foodstuff. For example when the enzyme is in
dry form it may be combined with an anticaking agent.
[0073] It will be appreciated by one skilled in the art that in the
practice of the present invention one contacts the foodstuff with a
sufficient amount of enzyme to prevent and/or reduce a acrylamide
formation and/or acrylamide precursor formation. Typical amounts of
enzyme which may be contacted with the foodstuff are from 0.05 to
50 U/g (units of enzyme per gram of foodstuff), from 0.05 to 10
U/g, from 0.05 to 5 U/g, from 0.05 to 3 U/g, from 0.05 to 2 U/g,
from 0.1 to 2 U/g, from 0.1 to 1.5 U/g, and from 0.5 to 1.5
U/g.
[0074] In one preferred aspect the use/process of the present
invention further comprises use of a catalase or contacting a
catalase with a foodstuff to remove oxygen and thereby prevent
and/or reduce acrylamide formation and/or acrylamide precursor
formation (such as 2-propenal formation).
[0075] In some aspects the foodstuff contains an amino acid. In
some aspects the amino acid is asparagine. It has been identified
that asparagine is particularly important in the formation of
acrylamide in foodstuffs.
[0076] In a preferred aspect the enzyme prevents and/or inhibits
Amadori reactions and subsequent reactions with asparagine
resulting in the formation of acrylamide.
[0077] In some aspects the foodstuff contains a protein. In some
aspects the foodstuff contains a peptide.
[0078] Acrylamide formation and/or acrylamide precursor formation
in a foodstuff may take place during the heating thereof or may
take place during storage of the foodstuff. For example acrylamide
formation and/or acrylamide precursor formation can happen upon
storage of any kind of seeds without heating. The enzyme of the
present invention, such as HOX, may still be useful however in
removing a second mole of aldose or ketose sugar which may react
with the already formed Amadori product to yield the diketoseamine
or diaminosugar.
[0079] Moreover the system of the present invention may prevent
loss of the nutritionally important Lysine in foods.
[0080] As a further addition it may be noted that reducing sugars
may play an important role in the initiation of Amadori and
Maillard reactions at certain moisture levels of the foodstuff
(8-12%), but that lipid auto-oxidation, which is also known to
initiate Amadori reactions, becomes increasingly common at low
moisture levels (6%) (McDonald 1999). Lipid oxidation may actually
be the primary cause for the initiation of Amadori or Maillard
reactions when reducing sugars are absent. The present enzyme, such
as HOX, may serve the dual purpose of removing both reducing sugars
and oxygen and thereby preventing lipid oxidation as well as sugar
hydrolysis at all moisture levels.
[0081] The present invention will now be described in further
detail by way of example only with reference to the accompanying
figures in which:--
BRIEF DESCRIPTION OF THE DRAWINGS
[0082] FIG. 1. Results from the use of hexose oxidase and glucose
oxidase to reduce the amount of acrylamide developed by frying
potato chips.
[0083] FIGS. 2 and 3. Results from the use of hexose oxidase and
glucose oxidase to reduce the amount of acrylamide developed by
baking potato chips.
[0084] FIG. 4. Statistical analysis of the results in FIG. 2.
[0085] FIG. 5. SRM Chromatograms of an extract of a fried potato
spiked with 1000 ng/15 ml [.sup.13C.sub.3]acrylamide (internal
standard). The transitions monitored are m/z 72>m/z 55 (upper,
acrylamide) and m/z 75>m/z 58 (lower,
[.sup.13C.sub.3]acrylamide).
[0086] FIG. 6. Pathways of formation of key flavour intermediates
and products in the Maillard reaction.
[0087] FIG. 7. Loss of lysine occurring as a result of heating of
several foods.
[0088] FIG. 8. Reaction pattern of the formation of melanoidins
from aldose sugars and amino compounds.
[0089] FIG. 9. Reversible formation of glycosylamines in the
browning reaction.
[0090] FIG. 10. Amadori rearrangement.
[0091] FIG. 11. Structure of
1-deoxy-1-glycino-.beta.-D-fructose.
[0092] FIG. 12. Heyns rearrangement.
[0093] FIG. 13. 1,2-enolization mechanism of the browning
reaction.
[0094] FIG. 14. Proposed browning reaction mechanism according to
Burton and McWeeney.
[0095] FIG. 15. Effect of temperature on the reaction rate of
D-glucose with DL-leucine.
[0096] FIG. 16. Effect of pH on the reaction rate of D-glucose with
DL-leucine.
[0097] FIG. 17. Decomposition of cysteine by a lipid free
radical.
DETAILED DESCRIPTION OF THE INVENTION
Examples
[0098] Acrylamide content of foodstuffs may be determined in
accordance with J Agric Food Chem. 2002 Aug. 14;
50(17):4998-5006.
Example 1
Pizza with Mozzarella Cheese
[0099] 20 g mozzarella cheese (Karoline's Dansk mozzarella, 25%
protein, 1% carbohydrate and 21% fat) is scaled in a beaker. 1 ml
Hexose Oxidase solution (7.5 HOX units/mi) is sprayed onto the
cheese. As a control 1 ml water is sprayed onto another sample of
mozzarella cheese. The cheese is stored for 2 hours at room
temperature. A dough is made from flour, salt and water. 10 g dough
is scaled and placed in a petri dish. 5 grams of mozzarella cheese
is placed on top of the dough and baked at 225.degree. C. for 7
min. Another sample is baked for 15 min. After baking the samples
are evaluated.
[0100] The samples in accordance with the present invention have a
lower content of acrylamide than the control samples.
Example 2
[0101] The effect of hexose oxidase is tested in a gratin made by
the following procedure.
[0102] 75 g shortening (mp. 35.degree. C.) and 100 g flour are
heated in a pot during mixing. 350 ml skim milk (preheated to
90.degree. C.) is added during continued mixing. Salt and pepper is
added. 4 eggs are divided into yolk and egg white. The egg yolks
are added individually. The egg white is whipped to a foam with 10
gram baking powder and mixed carefully into the dough. The dough is
placed in 2 aluminium trays. One of the trays is sprayed with a
solution of hexose oxidase 7.5 Units/ml and kept at room
temperature for 30 minutes. The gratin is then baked in a air
circulating oven at 175.degree. C. for 20 minutes. After baking the
gratin is evaluated.
[0103] The samples in accordance with the present invention have a
lower content of acrylamide than the control samples.
Example 3
[0104] The consumption of fried potato as French fries (pommes
frites) and potato chips (crisps) has increased significantly
during the past two decades. One of the important parameters in the
production of fried potatoes is level of reducing sugar. The level
should remain low, because high level of reducing sugar contribute
to higher levels of acrylamide.
[0105] In order to prevent an increase in the level of reducing
sugar in potatoes during storage potatoes are often sprayed with a
herbicide called chlorpropham, which prevents the potato from
sprouting. Sprouting induces amylases in the potato which in turn
form reducing sugars.
[0106] In this study one investigated if it is possible to reduce
levels of acrylamide in fried potatoes by adding HOX to sliced
potatoes before frying.
[0107] Procedure
[0108] Organic grown potatoes are used in order to ensure that no
herbicides has been used. The potatoes are peeled and sliced into 2
mm thick slices using a food processor. Half of the slices are
immersed in a water solution of HOX containing 100 Units/ml for 3
minutes. The other half of the potato slices are immersed in water
for 3 minutes. The slices are then stored in a closed container for
over night (16 hours) and then fried in vegetable oil for 2 minutes
at 180.degree. C.
[0109] Results
[0110] The samples in accordance with the present invention have a
lower content of acrylamide than the control samples.
Example 4
Crisp Bread with Rye Flour
[0111] 125 g rye flour
[0112] 125 g flour
[0113] 0.5 tsp baking powder
[0114] 3 tsp sugar
[0115] 2 tsp salt
[0116] 100 g margarine
[0117] 1.25 dl milk
[0118] 1 egg
[0119] Procedure [0120] Mix dry ingredients [0121] Crumble
margarine into mixture, and quickly knead the dough with water and
whisked egg [0122] Leave the dough to rest for 20 minutes, then
roll it out on the plate, prick and cut it into 8.times.20 cm big
loaves [0123] Bake for 10 minutes at 190.degree. C. until light
brown [0124] Break gently into pieces.
[0125] Results
[0126] The samples in accordance with the present invention have a
lower content of acrylamide than the control samples.
Example 5
Determination of Glucose Oxidase and Herose Oxidase Activity
[0127] Definition: 1 glucose oxidase (GOX) unit corresponds to the
amount of enzyme which under the specified conditions results in
the conversion of 1 .mu.mole glucose per minute, with resultant
generation of 1 .mu.mole of hydrogen peroxide (H.sub.2O.sub.2).
[0128] Definition: 1 hexose oxidase (HOX) unit corresponds to the
amount of enzyme which under the specified conditions results in
the conversion of 1 .mu.mole of glucose per minute, with resultant
generation of 1 .mu.mole of hydrogen peroxide (H.sub.2O.sub.2).
[0129] Assay of GOX and HOX activity in microtiter plates (300
.mu.l).
[0130] The commonly used horse radish peroxidase dye substrate ABTS
was incorporated into an assay, measuring the production of
H.sub.2O.sub.2 produced by HOX or GOX respectively. ABTS serves as
a chromogenic substrate for peroxidase. Peroxidase in combination
with H.sub.2O.sub.2 facilitates the electron transport from the
chromogenic dye, which is oxidised to an intensely green/blue
compound.
[0131] An assay mixture contained 266 .mu.l .beta.-D-glucose (Sigma
P-5504, 0.055 M in 0.1 M sodium phosphate buffer, pH 6.3), 11.6
.mu.l 2,2'-Azino-bis(3-ethylbenzothiozoline-6-Sulfonic
acid)(ABTS)(Sigma A-9941, 5 mg/ml aqueous solution), 11.6 .mu.l
peroxidase (POD)(Sigma P-6782, 0.1 mg/ml in 0.1 M sodium phosphate
buffer, pH 6.3) and 10 .mu.l enzyme (HOX or GOX) aqueous
solution.
[0132] The incubation was started by the addition of glucose at
25.degree. C. The absorbance was monitored at 405 nm in an ELISA
reader. A standard curve, based on varying concentrations of
H.sub.2O.sub.2, was used for calculation of enzyme activity
according to the definition above.
[0133] The reaction can be described in the following manner:
.beta.-D-glucose+O.sub.2+2H.sub.2O.fwdarw.gluconic
acid+2H.sub.2O.sub.2 (1)
H.sub.2O.sub.2+2ABTS (colorless)+2H.sup.+.fwdarw.2H.sub.2O+2ABTS
(blue/green) (2)
[0134] Reaction (1) is catalysed by enzyme (HOX or GOX)
[0135] Reaction (2) is catalysed by enzyme (POD)
Example 6
Use of Hexose Oxidase and Glucose Oxidase to Reduce the Amount of
Acrylamide Developed by Frying Potato Chips
[0136] Frying
[0137] Italian potatoes of the sort Nicola, were peeled and sliced
into pieces of approximately (3 mm.times.30 mm.times.40 mm).
Portions of approx. 30 g of sliced potatoes were treated with 40 mL
of one of the incubation solutions as described below. During
treatment it was made sure that all potatoes were covered with
solution and the incubating beakers were stirred at RT for 4 hours
in total.
[0138] After the enzyme treatment the potato slices where air dried
for app. 30 min and fried for 2.5 min in pure rapeseed oil
(175.degree. C.). Subsequently the potatoes were spread on tissue
paper and allowed to cool for approx. 30 min. They were stored dark
in closed containers at -20.degree. C. They were then purified and
analysed as described in example 7.2.
[0139] Treatment: [0140] (0) 40 mL of demineralised water
(=control) [0141] (1) 40 mL demineralised water containing 5 U/mL
glucose oxidase (GOX, Sigma G-6125) [0142] (2) 40 mL demineralised
water containing 5 U/mL hexose oxidase (HOX)
[0143] The results of the experiment are summarized in FIG. 1.
[0144] It is evident from FIG. 1 that incubation prior to frying,
using an incubation solution containing either GOX or HOX, had an
effect on the relative level of acrylamide found in the fried
potato. The largest effect was observed using HOX (.about.65%
reduction) (see treatment 2). A smaller effect was observed using
the same dosage of GOX (.about.41% reduction) (see treatment
1).
Example 7
[0145] Use of Hexose Oxidase and Glucose Oxidase to Reduce the
Amount of Acrylamide Developed by Baking Potato Chips.
[0146] 7.1. Baking
[0147] Italian potatoes of the sort Nicola, were peeled and sliced
as described in Example 6.
[0148] Portions of app 50 g were treated with 100 mL of incubation
solution and incubated for 15 min, while stirring at RT. During
treatment it was made sure that all potatoes were covered with
solution.
[0149] After the enzyme treatment the potato slices where air dried
for approx. 30 min and baked in a pre-heated oven for 30 min at
175.degree. C. To account for differences in heating conditions of
the oven, the baking plate was divided into 9 segments of equal
size. Potatoes treated as in (1)-(3) (see below), were divided into
9 equal fractions and 1 fraction from each was placed in each
segment to a total of 3 fractions per segment. This was done to
minimize the chance of faulty results as a consequence of uneven
heating in the oven. Subsequently the potatoes were spread on
tissue paper and allowed to cool for approx. 30 min. They were
stored dark in closed containers at -20.degree. C.
[0150] Treatment:
[0151] (1) No incubation
[0152] (2) 100 mL demineralised water
[0153] (3) 100 mL demineralised water containing 50 U/mL hexose
oxidase (HOX)
[0154] The results of the experiment are summarized in FIG. 2 and
FIG. 3.
[0155] Through statistical analysis of the results in FIG. 2, it
was found that HOX treated samples show significantly lower content
of acrylamide compared to water treated samples.
TABLE-US-00001 TABLE 1 Table of Least Squares Means for Amount with
95.0 Percent Confidence Intervals Stnd. Lower Upper Level Count
Mean Error Limit Limt GRAND MEAN 12 3147.75 F1 Blank 4 3148.0
272.466 2531.64 3764.36 HOX 4 2501.5 272.466 1885.14 3117.86 Water
4 3793.75 272.466 3177.39 4410.11
[0156] See FIG. 4
TABLE-US-00002 TABLE 2 Multiple Range Tests for Amount by FI
Method: 95.0 percent LSD Homogeneous F1 Count LS Mean LS Sigma
Groups HOX 4 2501.5 272.466 X Blank 4 3148.0 272.466 XX Water 4
3793.75 272.466 X Contrast Difference +/-Limits Blank - HOX 646.5
871.668 Blank - Water -645.75 871.668 HOX- Water *-1292.25 871.668
*denotes a statistically significant difference
[0157] 7.2 Sample Preparation and Quantification by LC-MS/MS
Experimental
Materials
[0158] Methanol (Lab Scan, Dublin, Ireland), acetic acid, reagent
grade ACS from Scharlau Chemie S.A. (Barcelona Spain).
[0159] Oasis MAX (6 cc, 150 mg, Part No. 186000370), Oasis MCX (6
cc, 150 mg, Part No. 186000256) from Waters (Milford, Mass.,
USA).
[0160] Acrylamide-1,2,3-.sup.13C.sub.3, 1 mg/ml methanol (Product
nr. CLM-813-1.2) from Cambridge Isotope Laboratories, Inc. (MA,
USA). Acrylamide (Product nr. 14857-1) from Aldrich, (Germany).
[0161] Instrumentals
[0162] The HPLC system consisted of a quaternary pump (G1311A),
autosampler (G1313A), column compartment (G1316A) all from Agilent
Technologies (Waldbronn, Germany).
[0163] An LCQ Deca Ion Trap mass spectrometer from Thermo Finnigan
(San Jose, Calif., USA).
[0164] Column (Atlantis.TM. dC.sub.18 3 .mu.m, 2.1 mm id.*150 mm)
from Waters (Milford, Mass., USA).
[0165] Chromnatographic and MS Conditions
[0166] Mobile Phase
[0167] H.sub.2O/MeOH/AcOH (1000/5/1 by volume)
[0168] The flow rate was 0.20 mimin.
[0169] MS Detector Settings
[0170] Capillary Temp (C): 275
[0171] Sheath Gas Flow: 96
[0172] Aux Gas Flow: 3
[0173] Source Type: ESI
[0174] Positive Mode
[0175] Source Voltage (kV): 2.00
[0176] MSn Micro Scans: 2
[0177] MSn Max Ion Time (ms): 500
[0178] Scan Event Details:
TABLE-US-00003 1: Pos (71.9) > (40.0-80.0) MS/MS: Amp. 34.0% Q
0.450 Time 30.0 IsoWidth 1.0 2: Pos (74.9) > (40.0-80.0) MS/MS:
Amp. 34.0% Q 0.450 Time 30.0 IsoWidth 1.0
[0179] Standard and Sample Preparation
[0180] Calibration standards (acrylamide) were prepared with the
following concentrations: 500, 150, 50, 15, 5 ng/ml in water. The
concentration of internal standard
(acrylamide-1,2,3-.sup.13C.sub.3) was maintained at 40 ng/ml.
[0181] The sample to be analysed was coarsely ground with a knife.
An aliquot (1 g) was homogenized (Ultra-Turrax T25) with 15 ml of
internal standard, (ISTD, 1000 ng acrylamide
1,2,3-.sup.13C.sub.3/15 ml H.sub.2O) in a 100 ml beaker.
[0182] The homogenate was transferred to a 50 ml centrifuge tube
and 2 ml of dichloromethane were added. The mixture was shaken and
centrifuged at 18000 rev/min (=25000 RCF) in a Sorvall RC-5B
centrifuge for 20 min. at 4.degree. C.
[0183] An Oasis MAX cartridge and an Oasis MCX cartridge were each
conditioned with 5 ml methanol followed by 2*5 ml water. After
conditioning, they were combined in series with Oasis MAX on
top.
[0184] An aliquot (1.5 ml) of the supernatant (water) was passed
through the Oasis MAX/Oasis MCX tandem (fraction 1).
[0185] Water (5 ml) was added to the Oasis MAX/Oasis MCX tandem and
the eluent was collected in three fractions: Fraction 2 (1 ml),
fraction 3 (2 ml) and fraction 4 (2 ml). Fraction 3 was filtered
through a 0.45-.mu.m filter (13 mm GHP 0.45 .mu.m Minispike,
Waters) and subjected to analysis.
[0186] Appendix 1 and Appendix 2 follow.
APPENDIX 1
Chemical Mechanisms for Acrylamide Formation
[0187] Food science and technology have had interest in acrylamide
itself (and/or its derivatives incl. polymers), and its
applications and possible toxic effects for many years. For
example, there are many reports on can coatings and food packaging,
on food additives (preservatives, artificial sweeteners etc.) and
on acrylamide polymers of suitable quality with low residual
acrylamide monomer levels that are used in, e.g. the U.S. for
treatment of poultry, potato, corn, and other wastes, with the
resulting concentrated solids used as components of blended animal
feeds (14-19).
[0188] There are only a few earlier reports on the occurrence of
acrylamide in foods. For example, acrylamide has been reported to
be present in plant material (potatoes, carrots, radish, lettuce,
Chinese cabbage, parsley, onions, spinach, and rice paddy) (20). In
1 g plant samples, 1.5.100 ng acrylamide could be detected.
Acrylamide was also reported to occur in sugar (21). The origin of
the detected acrylamide in these foods is not known. It might be
exogenous.
[0189] To the best of our knowledge, no proposed or proven reaction
routes for the formation of acrylamide during food processing have
been published. Therefore, what are described below are the
hypotheses we find most relevant and probable in a food processing
situation. [0190] A. Acrolein (2-propenal, CH2=CH--CHO) is a three
carbon aldehyde and thus reminds the structure of acrylamide
(CH2=CH--C(O)--NH2). Further, acrolein is known to be formed by:
[0191] 1. transformation of lipids [0192] 2. degradation of amino
acids and proteins [0193] 3. degradation of carbohydrates [0194] 4.
the Maillard reaction between amino acids or proteins and
carbohydrates
[0195] Therefore, acrolein is a very probable precursor of
acrylamide. Simple, fundamental chemical transformations (such as
reaction with ammonia liberated from amino acids) can then convert
acrolein (or a derivative from it) into acrylamide. The production
of acrylamide through the reaction of acrolein with ammonia has
been demonstrated in model systems (22). [0196] B. Alternative
formation mechanisms of acrylamide do not necessarily involve
acrolein. For example, proteins and/or amino acids can after a
series of transformations, such as hydrolyses, rearrangements,
decarboxylations etc., eventually lead to acrylamide.
[0197] The processes mentioned above (A and B) are complicated and
involve multistage reaction mechanisms which might also include
free radical reactions to acrolein or acrylamide (23-25).
Acrolein Formation from Lipids
[0198] When oil is heated at temperatures above the smoke point,
glycerol is degraded to acrolein, the unpleasant acrid black and
irritating smoke (26-29). The formation of acrolein is known to
increase with the increase in unsaturation in the oil and to lead
to a lowering of the smoke point. The smoke point is higher for
oils with higher content of saturated fatty acids and lower content
of polyunsaturated acids. The smoke points for some of the main
oils and fats are as follows: palm 240.degree. C., peanut
220.degree. C., olive: 210.degree. C., lard and copra 180.degree.
C., sunflower and soybean 170.degree. C., corn 160.degree. C.,
margarine 150.degree. C. and butter 110.degree. C. Usually the
smoke starts to appear on the surface of heated oils before their
temperature reaches 175.degree. C. The oil is first hydrolyzed into
glycerol and fatty acids and then acrolein is produced by the
elimination of water from glycerol by a heterolytic acid-catalyzed
carbonium ion mechanism followed by oxidation (30).
##STR00003##
[0199] Besides the above-mentioned mechanism for the formation of
acrolein from acylglycerols, acrolein can also be produced as a
result of oxidation of polyunsaturated fatty acids and their
degradation products (31-34). A number of aldehydic products
(including malondialdehyde, C3-C10 straight chain aldehydes, and
.alpha.,.beta.-unsaturated aldehydes, such as 4-hydroxynonenal and
acrolein) are known to form as secondary oxidation products of
lipids (35). Acrolein was also found to form in vivo by the
metal-catalyzed oxidation of polyunsaturated fatty acids including
arachidonic acid (36).
Acrolein Formation from Amino Acids, Proteins and Carbohydrates
[0200] Several sources for the formation of acrolein are known. It
may arise from degradation of amino acids and proteins (37, 38),
from degradation of carbohydrates (39), and from the Maillard
reaction between amino acids or proteins and carbohydrates (40,
41). Many possible routes for the formation of this three-carbon
aldehyde--taking the starting point from many different sugars or
amino acids--may be proposed. Its formation from methionine by the
Strecker degradation in the frame of the Maillard reaction is one
example. Alanine, with its tree-carbon skeleton, has also been
suggested as a possible source. However, fission reactions of
longer carbon chains are common and well-known, so at present there
is no basis to give priority to any specific reaction routes.
Formation of Acrylamide Through Amino Acid Reactions not Involving
Acrolein
[0201] There are also numerous, plausible reaction routes by which
amino acids (or proteins) may form acrylamide without going through
acrolein. Within the frame of complex, multistage reaction
mechanisms, involving hydrolyses, rearrangements, decarboxylations,
deaminations etc., many specific mechanistic pathways may be
suggested. Decarboxylation and deamination of aspargine, and
transformations of dehydroalanine (formed from e.g. serine or
cysteine) are some examples of reaction routes that have been
proposed. But also in this case these can only be seen as possible
examples, and similarly to above, there is no basis to give
priority to any specific routes.
Conclusion
[0202] Since no systematic studies have been performed or reported,
there is at present no evidence to point out any specific reaction
routes for acrylamide formation, or to exclude any possibilities.
Most probably a multitude of reaction mechanisms is involved,
depending on food composition and processing conditions.
Further Reactions of Formed Acrolein and Acrylamide
[0203] As mentioned above, acrolein can be converted into
acrylamide by a series of fundamental reactions. However, both
acrolein and acrylamide are reactive, because of their double bonds
and the amino group of acrylamide. They can readily react further
with other reactive groups present in the food matrix or formed
during the heating process. For example, acrylamide can react with
small reactive molecules, such as urea (CO(NH2).sub.2) and
formaldehyde (HCHO), or with glyoxal ((CHO)2), aldehydes (RCHO),
amines (R2NH), thiols (RSH) etc. Furthermore, the products shown in
the following scheme can even react further in the same mode of
reaction.
##STR00004##
[0204] These types of reactive functional groups may also be found
in macromolecules, such as proteins, for instance. (Cf. adduct
formation with valine in the globin chain of hemoglobin described
above. In hemoglobin adducts are formed not only with valine, but
also with e.g. cystein.) The presence or absence of reactive groups
(or its concentration) in the food matrix may thus be one
explanation of differences in final acrylamide content in different
food systems. The resulting acrylamide level may be due to a
balance between formation and further reactions. The low acrylamide
levels in heated meat products could, for instance, depend on
adduct formation between acrylamide (or acrolein) and proteins.
Factors with Possible Influence on Acrylamide Formation
[0205] A couple of different chemical mechanisms for the formation
of acrylamide have been outlined above. Obviously, as long as the
mechanism or mechanisms are not confirmed, the influencing factors
can not be established. Thus, what is presented here are attempts
to identify what factors would be of importance (regarding
processing conditions or product composition) if a specific
reaction route is the prevailing one. Specific emphasis is put on
the Maillard reaction, since this reaction system involves many of
the basic carbohydrate and amino acid reactions. Another major
reaction in foods during processing, which could be of importance,
is lipid hydrolysis followed by oxidation of the fatty acids.
Acrolein Formation from Lipids
[0206] Acrolein may be formed from the glycerol part of
triglycerides or through oxidation of fatty acids. This means that
factors favouring lipid hydrolysis as well as factors favouring
lipid oxidation would promote acrolein formation. Temperature is an
important factor for both these reactions. Regarding hydrolysis, pH
may also be of importance and high as well as low pH may be
supposed to favour acrolein formation. Regarding oxidation, lipid
composition is of key importance; the higher the degree of
unsaturation, the lower the stability. Protection against oxygen
and light will limit the oxidation and prooxidants, such as metals,
should be avoided. The protective effect of antioxidants should
also be taken into account.
The Maillard Reaction as the Route for Acrylamide Formation
[0207] The Maillard reaction has been proposed as a route for
acrolein formation. Also the direct formation of acrylamide through
amino acid transformations has been proposed. These amino acid
transformations also involve reactions common in the Maillard
reaction system.
Maillard Reaction Basics
[0208] The Maillard reaction (MR) is one of the most important
chemical reactions in food processing, with influence on several
aspects of food quality. Flavour, colour and nutritional value may
be affected and certain reaction products have been noticed to be
antioxidative, antimicrobial, genotoxic etc. The practical
applications of Maillard chemistry in food processing are,
therefore, a matter of balance between favourable and unfavourable
effects, and the aim of the food manufacturer is to find an optimum
in this balance. This may be accomplished by influencing the main
variables affecting the MR (42).
[0209] The Maillard reaction takes place in 3 major stages and is
dependent upon factors, such as concentrations of reactants and
reactant type, pH, time, temperature, and water activity. Free
radicals and antioxidants are also involved (43).
[0210] The early stage (step 1) involves the condensation of a free
amino group (from free amino acids and/or proteins) with a reducing
sugar to form Amadori or Heyns rearrangement products. The advanced
stage (step 2) means degradation of the Amadori or Heyns
rearrangement products via different alternative routes involving
deoxyosones, fission or Strecker degradation. A complex series of
reactions including dehydration, elimination, cyclization, fission
and fragmentation result in a pool of flavour intermediates and
flavour compounds. Following the degradation pathway as illustrated
schematically in FIG. 6, key intermediates and flavour chemicals
can be identified.
[0211] One of the most important pathways is the Strecker
degradation in which amino acids react with dicarbonyls (formed by
the Maillard reaction) to generate a wealth of reactive
intermediates. Typical Strecker degradation products are aldehydes,
e.g. formaldehyde, acetaldehyde, and possibly propenaldehyde
(acrolein). Strecker degradation results in degradation of amino
acids to aldehydes, ammonia and carbon dioxide (44) and takes place
in foods at higher concentrations of free amino acids and under
more drastic reactions, e.g. at higher temperatures or under
pressure (45). Pathways of formation of key flavour intermediates
and products in the Maillard reaction (43) are shown in FIG. 6.
[0212] The final stage (stage 3) of the MR is characterized by the
formation of brown nitrogenous polymers and co-polymers. While the
development of colour is an important feature of the reaction,
relatively little is known about the chemical nature of the
compounds responsible. Colour compounds can be grouped into two
general classes--low molecular weight colour compounds, which
comprise two to four linked rings, and the melanoidins, which have
much higher molecular weights.
Review of Factors Influencing the Maillard Reaction
[0213] Factors that are particularly important for the MR are the
starting reactants, e.g. type of sugar and amino acid (protein),
time, temperature and water activity. Presence of metal salts
(pro-oxidants), and inhibitors, like antioxidants and sulphite,
might all have an impact.
Starting Reactants--Reducing Sugar and Amino Acids/Proteins
[0214] MR requires reducing sugars, i.e. sugars containing keto- or
aldehydes (free carbonyl groups). The reactivity of different
sugars can be summarised in the following way (46): [0215] The
shorter carbon chain, the sugar has, the greater are the lysine
losses (MR). [0216] Pentoses are more reactive than hexoses and
disaccharides in yielding brown colour. [0217] Aldoses are more
reactive than ketoses both in aqueous solution model systems and at
storage (low water content) [0218] Among isomeric sugars,
stereochemistry is important. Thus ribose is more reactive than
xylose monitored as lysine losses.
[0219] All monosacharides are reducing sugars. (Sugar alcohols do
not participate in MR.) Among the disaccharides all sugars except
sucrose are reducing sugars. In oligosaccharides and starch only
the end-terminal monosaccharide is a reducing sugar. Starch and
sugars, such as sucrose, lactose, maltose etc can easily hydrolyse
upon heating above 100.degree. C. at slightly acidic pH, resulting
in the formation of monosaccharides (reducing sugars). Thus,
thermal processing often result in a continuous supply of reducing
sugar formed from complex carbohydrates.
[0220] Most studies concerning reactivity of amino acids have been
performed on free amino acids in diluted aqueous solutions. The
reactivity among the diamino acids increased with the length of the
carbon chain. Among the amino acids studied lysine was most
reactive. In proteins and peptides, only free amino groups can
react, i.e. N-terminal a-amino groups and -amino groups.
Temperature and Time
[0221] The temperature dependence of chemical reactions is often
expressed as the activation energy, Ea, in the Arrhenius equation.
The higher the value of Ea, the more temperature dependent is the
reaction rate. Activation energy data for the MR have been reported
within a wide range, 10-160 kJ/mole, depending on, among other
things, water activity and pH and what effect of the reaction has
been measured. The temperature dependence of the MR is also
influenced by the participating reactants. The temperature effect
is also affected by the other variables and different aspects of
the MR thus differ in temperature dependence (42).
Water
[0222] Water has both an inhibitory and an accelerating impact on
the MR. Water acts partly as a reactant and partly as a solvent and
transporting medium of reactants (reactant mobility). In the
initial steps of the MR, 3 moles of water are formed per mol
carbohydrate. Thus the reaction occurs less readily in foods with a
high aw value. Water might depress the initial glucosylamine
reaction, but enhance the deamination step later in the
reaction.
[0223] The results from studies in model systems for optimal water
concentration or water activity (free water) or relative humidity
(RH) vary markedly depending on selected reactants and how the MR
is evaluated--as loss in lysine or browning intensity. Several
studies have been performed of which most claim the max aw to be
between 0.3 and 0.7 (47). However, most data on the aw influence
are based on studies at relatively low temperatures (30-60.degree.
C.). At higher temperature, more relevant to heat processes,
considerably lower aw has been shown to be favourable to the MR
(42).
[0224] The main explanation to an optimum reaction rate at an
intermediate aw is that the reactants are diluted at the higher aw,
while at a lower aw the mobility of reactants is limited, despite
their presence at increased concentrations.
pH
[0225] The MR itself has a strong influence on pH. Therefore,
aqueous model systems based on reflux boiling of sugars and amino
acids need to be buffered since the pH quickly drops from 7 to 5.
Low pH values (<7) favour the formation of furfurals (from
Amadori rearrangement products), while the routes for reductones
and fission products are preferred at a high pH.
[0226] However, the overall effect of pH is not clear cut, since
the reactions take place by all the three pathways. In unbuffered
water solutions, pH decrease during MR and buffering with alkali
has a catalytic effect.
[0227] Reactivity of different amino acids at various pHs has been
studied. Browning of a glucose solution upon heating was obtained
first when pH exceeded 5 and it increased with increasing pH. The
degree of browning varied with the position of the amino group. The
function of pH is linked with specific reaction steps of the MR.
Initially only non-protonised forms of amino acids a can form
Schiff's base. This explains the pronounced changes in reactivity
(monitored as browning) which happens when pH passes the
isoelectric point of the amino group in the reacting amino acid.
Thus, optimal pH for the MR varies with the system used and how the
reaction is monitored (e.g. lysine losses or browning).
Inhibition of the Maillard Reaction
[0228] Measures to inhibit the Maillard reaction in cases where it
is undesirable, involve lowering of the pH value, maintenance of
lowest possible temperatures and avoidance of critical water
contents (moistures below 30%, during processing and storage), use
of non-reducing sugars, and addition of sulphite (45). The use of
the inhibitor, sulphur dioxide, constitutes an important way of
controlling the Maillard reaction. It may combine with early
intermediates. However, sulphite only delays colour formation and
it is interesting to note that the colour formed in
sulphite-treated systems is less red and more yellow than in
untreated systems.
Maillard Reactions and Food Processing
[0229] In exploiting the Maillard reaction, the key target for the
food industry is to understand and harness the reaction pathways
enabling improvement of existing products and the development of
new products. While it would be easy to assume that this means the
generation of flavour and colour, not all Maillard products endow
positive characteristics to foods and ingredients. The positive
contributions of the MR are flavour generation and colour
development. The negative aspects are off-flavour development,
flavour loss, discoloration, loss of nutritional value and
formation of toxic Maillard reaction products (MRPs). In applying
the MR, there are challenges that are common to the food industry,
independent of the type of the product. These challenges can be
classified as follows: maintenance of raw material quality;
maintenance of controlled processes for food production;
maintenance of product quality; extension of product shelf-life
(42, 43).
Flavour/Aroma
[0230] The most common route for formation of flavours via the MR
comprises the interaction of a-dicarbonyl compounds (intermediate
products in the MR, stage 2) with amino acids through the Strecker
degradation reactions. Alkyl pyrazines and Strecker aldehydes
belong to commonly found flavour compounds from MR. For example,
low levels of pyrazines are formed during the processing of potato
flakes when the temperature is less than 130.degree. C., but
increases tenfold when the temperature is increased to 160.degree.
C., and decreases at 190.degree. C., probably due to evaporation or
binding to macromolecules. The aroma profile varies with the
temperature and the time of heating. At any given temperature-time
combination, a unique aroma, which is not likely to be produced at
any other combination of heating conditions, is produced.
Temperature also affects the development of aroma during extrusion
cooking.
Colour
[0231] The coloured products of the Maillard reaction are of two
types: the high molecular weight macromolecule materials commonly
referred to as the melanoidines, and the low molecular weight
coloured compounds, containing two or three heterocyclic rings
(48). Colour development increases with increasing temperature,
with time of heating, with increasing pH and by intermediate
moisture content (aw=0.3-0.7). Generally, browning occurs slowly in
dry systems at low temperatures and is relatively slow in
high-moisture foods. Colour generation is enhanced at pH>7. Of
the two starting reactants, the concentration of reducing sugar has
the greatest impact on colour development. Of all the amino acids,
lysine gives the largest contribution to colour formation and
cysteine has the least effect on colour formation.
Antioxidative Capacity
[0232] There are several reports on the formation of antioxidative
MRPs in food processing. The addition of amino acids or glucose to
cookie dough has been shown to improve oxidative stability during
the storage of the cookies. Heat-treatment of milk product prior to
spray drying has been reported to improve storage stability as has
heat treatments of cereals (42).
[0233] The antioxidant effect of the MRP has been extensively
investigated (49). It has been reported that the intermediate
reductone compounds of MRP could break the radical chain by
donation of a hydrogen atom: MRP was also observed to have
metal-chelating properties and retard lipid peroxidation.
Melanoidines have also been reported to be powerful scavengers of
reactive oxygen species (50). Recently, it was suggested that the
antioxidant activity of xylose-lysine MRPs may be attributed to the
combined effect of reducing power, hydrogen atom donation and
scavenging of reactive oxygen species (51).
Nutritive Value
[0234] Loss in protein quality is often associated with the MR,
especially in cereal products and milk powder produced by
heat-treatment. Usually the essential amino acid having an extra
free amino group, e.g. lysine, is most vulnerable. If the essential
amino acid also is the nutritionally limiting amino acid, the
influence of MR on the protein quality is substantial. This is not
a problem in cooking meat and fish, since these food items are very
rich in protein. Loss of protein quality in terms of nutritional
value is a more serious problems for heat-treatment and dehydration
of especially cereals, milk and their mixtures (breakfast cereals,
gruels, bread, biscuits), since carbohydrates dominates over
proteins in these food items and the proteins levels are also
generally low.
Toxic Effects
[0235] The possibilities that MPR could be mutagenic and/or
carcinogenic were explored with Ames test, around 20-25 years ago.
In general weak genotoxicity/mutagenic activities were found for
known MPRs. Most attention over the past decades has been paid on
the food mutagens found in the crust from cooked meat and fish.
Chemically, these compounds belong to a class of heterocyclic
amines, currently amounting to around 20 different species. Most of
them have been classified as possible food carcinogens (group 2B)
according to the International Agency for Research in Cancer (IARC)
based on long-term studies on rodents. The precursors of the
heterocyclic amines are free amino acids and for more than half of
the 20 species, also creatine (a natural energy metabolite present
in muscle cells only). Reducing sugars up to equimolar amounts
compared with amino acids and/or creatine enhance the yields of
heterocyclic amines markedly.
[0236] Thus MR and/or pyrolysis have been claimed to be important
mechanisms for the formation of these heterocyclic amines, where
Strecker aldehydes, pyrazines or pyridines and creatine have been
suggested to play an important role. The yields of these food borne
carcinogens are increasing with time and temperature, especially
from 150.degree. C. and above. The highest concentrations of
heterocyclic amines are found in the crust of pan-fried, grilled or
barbecued meat and fish. In addition, gravies prepared from dried
meat-juice collected from pan-residues or oven-roasting could be
rich in heterocyclic amines. Pro-oxidants, water activity in the
optimal range for the MR, and high temperatures (200-400.degree.
C.) enhance their yield. The average daily exposure for
heterocyclic amines is around 0.5 .mu.g/day and person, with a
range between 0-20 .mu.g. Antioxidants, excess of carbohydrates,
cooking temperatures below 200.degree. C. and moisture contents
above 30% reduce the occurrence of heterocyclic amines. Moreover,
heterocyclic amines rarely occur in plant foods even during
well-done cooking (52).
[0237] There is to our knowledge no report in the literature yet
concerning acrylamide formation linked with the MR.
APPENDIX 2
Nonenzymic Browning
[0238] The nonenzymic browning or Maillard reaction is of great
importance in food manufacturing and its results can be either
desirable or undesirable. An example of the first kind is the brown
crust formation on bread and one of the second kind is the brown
discoloration of evaporated and sterilized milk. In products in
which the browning reaction is favorable, the resulting color and
flavor characteristics are generally experienced as pleasant. In
other products, color and flavor become quite unpleasant.
[0239] The browning reaction can be defined as the sequence of
events which begins with the reaction of the amino group of amino
acids, peptides or proteins with a glycosidic hydroxyl group of
sugars and terminates with the formation of brown nitrogenous
polymers or melanoidins.
[0240] The reaction velocity and pattern are influenced in the
first place by the nature of the reacting amino acid or protein and
of the carbohydrate. This means that each kind of food may show a
different browning pattern. Generally, lysine is the most reactive
amino acid because of the free .epsilon.-amino group. Since lysine
is the limiting essential amino acid in many food proteins, its
destruction is of vital importance and can result in substantial
reduction of the nutritional value of the protein. Foods which are
rich in reducing sugars are very reactive, and this explains that
lysine in milk is destroyed more easily than in other foods (FIG.
7). Other factors which influence the browning reaction are:
temperature, pH, moisture level, oxygen, metals, phosphates, sulfur
dioxide and other inhibitors.
[0241] The browning reaction involves a number of steps and an
outline of the total pathway of melanoidin formation has been given
by Hodge (1953) shown in FIG. 8. According to Hurst (1972) five
steps are involved in the process: [0242] 1. The production of an
N-substituted glycosylamine from an aldose or ketose reacting with
a primary amino group of an amino acid, peptide or protein. [0243]
2. Rearrangement of the glycosylamine by an Amadori rearrangement
type of reaction to yield an aldoseamine or ketoseamine. [0244] 3.
A second rearrangement of the ketoseamine with a second mole of
aldose to result in the formation of a diketoseamine, or the
reaction of an aldoseamine with a second mole of amino to yield a
diamino sugar. [0245] 4. Degradation of the amino sugars with loss
of one or more molecules of water to give amino or nonamino
compounds. [0246] 5. Condensation of the compound formed in step 4
with each other or with amino compounds with formation of brown
pigments and polymers.
[0247] The formation of glycosylamines from the reaction of amino
groups and sugars is reversible (FIG. 9) and the equilibrium is
highly dependent on the moisture level present. The mechanism as
shown is thought to involve addition of the amine to the carbonyl
group of the open-chain form of the sugar, elimination of a
molecule of water, and closure of the ring. The rate is high at low
water content and this explains the ease of browning in dried and
concentrated foods.
[0248] The Amadori rearrangement of the glycosylamines involves the
presence of an acid catalyst and leads to the formation of
ketoseamine or 1-amino-1-deoxyketose according to the scheme of
FIG. 10. In the reaction of D-glucose with glycine the amino acid
reacts as the catalyst and the compound produced is
1-deoxy-1-glycino-.beta.-D-fructose (FIG. 11). The ketoseamines are
relatively stable compounds which are formed in maximum yield in
systems with 18% water content (Shallenberger and Birch 1975). A
second type of rearrangement reaction is the Heyns rearrangement
which is an alternative to the Amadori rearrangement and leads to
the same type of transformation. The mechanism of the Amadori
rearrangement (FIG. 10) involves protonation of the nitrogen atom
at carbon 1. The Heyns rearrangement (FIG. 12) involves protonation
of the oxygen at carbon 6.
[0249] Secondary reactions lead to the formation of diketoseamines
and diamino sugars. The formation of these compounds involves
complex reactions and in contrast to the formation of the primary
products does not occur on a mole for mole basis.
[0250] In the following step, the ketoseamines are decomposed by
1,2-enolization or 2,3-enolization. The former pathway appears to
be the more important one in the formation of brown color whereas
the latter results in the formation of flavor products. According
to Hurst (1972), the 1,2-enolization pathway appears to be the main
one leading to browning but also contributes to formation of
off-flavors through hydroxymethylfurfural, which may be a factor in
causing the off-flavors in stored, overheated or dehydrated food
products. The mechanism of this reaction is shown in FIG. 13 (Hurst
1972). The ketoseamine (1) is protonated in acid medium to yield
(2). This is changed in a reversible reaction into the
1,2-enolamine (3) and this is assisted by the N substituent on
carbon No. 1. The following steps involve the .beta.-elimination of
the hydroxyl group on carbon No. 3. In (4) the enolamine is in the
free base form and converts to the Schiff base (5). The Schiff base
may undergo hydrolysis and form the enolform (7) of 3-deoxyosulose
(8). In another step the Schiff base (5) may lose a proton and the
hydroxyl from carbon No. 4 to yield a new Schiff base (6). Both
this compound and the 3-deoxyosulose may be transformed into an
unsaturated osulose (9), and by elimination of a proton and a
hydroxyl group, hydroxymethylfurfural (10) is formed.
[0251] Following the production of 1,2-enol forms of aldose and
ketose amines, a series of degradations and condensations results
in the formation of melanoidins. The .alpha.-.beta.-dicarbonyl
compounds enter into aldol type condensations which lead to the
formation of polymers, initially of small size, highly hydrated and
in colloidal form. These initial products of condensation are
fluorescent and continuation of the reaction results in the
formation of the brown melanoidins. These polymers are of
non-distinct composition and contain varying levels of nitrogen.
The composition varies with the nature of the reaction partners,
pH, temperature and other conditions.
[0252] The flavors produced by the Maillard reaction also vary
widely. In some cases, the flavor is reminiscent of carmelization.
An important reaction contributing to the formation of flavor
compounds is the Strecker degradation of .alpha.-amino acids. The
dicarbonyl compounds formed in the previously described schemes
react in the following manner with .alpha.-amino acids:
TABLE-US-00004 ##STR00005## AROMA AND STRUCTURE CLASSIFICATION OF
BROWNED FLAVOR COMPOUNDS Aromas: Burnt Variable (pungent,
empyreumatic) (aldehydic, ketonic) Structures: ##STR00006##
Monocarbonyls (R--CHO, R--C:O--CH.sub.3) Examples of Glyoxal
Strecker compounds: aldehydes Pyruvaldehyde Isobutyric Diacteyl
Isovaleric Mesoxalic dialdehyde Methional Acrolein 2-Furaldehydes
2-Pyrrole aldehydes Crotonaldehyde C.sub.3--C.sub.6 Methyl ketones
Source: Hodge et al. (1972).
[0253] The amino acid is converted into an aldehyde with one less
carbon atom (Schonberg and Moubacher 1952). Some of the compounds
of browning flavor have been described by Hodge et al. (1972).
Corny, nutty, bready and crackery-aroma compounds consist of planar
unsaturated heterocyclic compounds with one or two nitrogen atoms
in the ring. Other important members of this group are partially
saturated N-heterocyclics with alkyl or acetyl group substituents.
Compounds that contribute to pungent, burnt aromas are listed in
Table 3. These are mostly vicinal polycarbonyl compounds and
.alpha.,.beta.-unsaturated aldehydes. They condense rapidly to form
melanoidins. The Strecker degradation aldehydes contribute to the
aroma of bread, peanuts, cocoa and other roasted foods. Although
acetic, phenylacetic, isobutyric and isovaleric aldehydes are
prominent in the aromas of bread, malt, peanuts and cocoa, they are
not really characteristic of these foods (Hodge et al. 1972).
[0254] A somewhat different mechanism for the browning reaction has
been proposed by Burton and McWeeney (1964) and is shown in FIG.
14. After formation of the aldosylamine, dehydration reactions
result in the production of 4- to 6-membered ring compounds. When
the reaction proceeds under conditions of moderate heating,
fluorescent nitrogenous compounds are formed and these react
rapidly with glycine to yield melanoidins.
[0255] The influence of reaction components and reaction conditions
results in a wide variety of reaction patterns. Many of these
conditions are interdependent. Increasing temperature results in a
rapidly increasing rate of browning, and not only reaction rate,
but also the pattern of the reaction may change with temperature.
In model systems, the rate of browning increases 2-3 times for each
10.degree. rise in temperature. In foods containing fructose, the
increase may be 5 to 10 times for each 10.degree. rise. At high
sugar contents, the rate may be even more rapid. Temperature also
affects the composition of the pigment formed. At higher
temperatures, the carbon content of the pigment increases and more
pigment is formed per mole of carbon dioxide released. Color
intensity of the pigment increases with increasing temperature. The
effect of temperature on the reaction rate of D-glucose with
DL-leucine is demonstrated in FIG. 15.
[0256] In the Maillard reaction, the basic amino group disappears
and, therefore, the initial pH or the presence of a buffer has an
important effect on the reaction. The browning reaction is slowed
down by decreasing pH, and the browning reaction can be said to be
self-inhibitory since the pH decreases with the loss of the basic
amino group. The effect of pH on the reaction rate of D-glucose
with DL-leucine is demonstrated in FIG. 16. The effect of pH on the
browning reaction is highly dependent on moisture content. When a
large amount of water is present, most of the browning is caused by
caramelization, but at low water levels and at pH greater than 6,
the Maillard reaction is predominant.
[0257] The nature of the sugars in a nonenzymic browning reaction
determines their reactivity. Reactivity is related to their
conformational stability or to the amount of open-chain structure
present in solution. Pentoses are more reactive than hexoses, and
hexoses more than reducing disaccharides. Nonreducing disaccharides
only react after hydrolsys has taken place. The order of reactivity
of some of the aldohexoses is mannose>galactose>glucose.
[0258] The effect of the type of amino acid can be summarized as
follows. In the .alpha.-amino acid series, glycine is the most
reactive. Longer and more complex substituent groups reduce the
rate of browning. In the co-amino acid series, browning rate
increases with increasing chain length. Ornithine browns more
rapidly than lysine. When the reactant is a protein, particular
sites in the molecule may react faster than others. In proteins,
the .epsilon.-amino group of lysine is particularly vulnerable to
attack by aldoses and ketoses.
[0259] Methods of preventing browning could consist of measures
intended to slow reaction rates, such as control of moisture,
temperature or pH or removal of an active intermediate. Generally,
it is easier to use an inhibitor. One of the most effective
inhibitors of browning is sulfur dioxide. The action of sulfur
dioxide is unique and no other suitable inhibitor has been found.
It is known that sulfite can combine with the carbonyl group of an
aldose to give an addition compound:
NaHSO.sub.3+RCHO.fwdarw.RCHOHSO.sub.3Na
but this reaction cannot possibly account for the inhibitory effect
of sulfite. It is thought that sulfur dioxide reacts with the
degradation products of the amino sugars which prevents these
compounds from condensation into melanoidins. A serious drawback of
the use of sulfur dioxide is that it reacts with thiamine and
proteins, thereby reducing the nutritional value of foods. Sulfur
dioxide destroys thiamine and is, therefore, not permitted for use
in foods containing this vitamin.
Chemical Changes
[0260] During processing and storage, a number of chemical changes
may occur in food proteins, some of which are desirable, others
undesirable. Such chemical changes may lead to compounds which are
non-hydrolyzable by intestinal enzymes or to modification of the
peptide side chains which render certain amino acids unavailable.
Mild heat treatments in the presence of water can significantly
improve the nutritional value in some cases. Sulfur-containing
amino acids may become more available and certain antinutritional
factors such as the trypsin inhibitors of soybeans may be
deactivated. Excessive heat in the absence of water can be
detrimental to protein quality, e.g., in fish proteins tryptophan,
arginine, methionine and lysine may be damaged. A number of
chemical reactions may take place during heat treatment including
decomposition, dehydration of serine and threonine, loss of sulfur
from cysteine, oxidation of cysteme and methionine, cyclization of
glutamic and aspartic acid and threonine (Mauron 1970).
[0261] One of the most important changes resulting in decomposition
of certain amino acids is the non-enzymic browning reaction or
Maillard reaction. For this reaction, the presence of a reducing
sugar is required. Heat damage may also occur in the absence of
sugars. Bjarnason and Carpenter (1970) demonstrated that heating of
bovine plasma albumin for 27 hours at 115.degree. C. resulted in a
50% loss of cystine and 4% of lysine. These authors suggest that
amide type bonds are formed by reaction between the .epsilon.-amino
group of lysine and the amide groups of asparagine or glutamine,
with the reacting units present either in the same peptide chain or
in neighboring ones.
[0262] Some amino acids may be oxidized by reacting with free
radicals formed by lipid oxidation. Methionine can react with a
lipid peroxide to yield methionine sulfoxide. Cysteine can be
decomposed by a lipid free radical according to the following
scheme in FIG. 17.
[0263] All publications mentioned in the above specification are
herein incorporated by reference. Various modifications and
variations of the described methods and system of the invention
will be apparent to those skilled in the art without departing from
the scope and spirit of the invention. Although the invention has
been described in connection with specific preferred embodiments,
it should be understood that the invention as claimed should not be
unduly limited to such specific embodiments. Indeed, various
modifications of the described modes for carrying out the invention
which are obvious to those skilled in chemistry or related fields
are intended to be within the scope of the following claims.
* * * * *