Note: Descriptions are shown in the official language in which they were submitted.
CA 02618225 2008-01-18
METHOD FOR REDUCING ACRYLAMIDE FORMATION
TECHNICAL FIELD:
The present invention relates to a method for reducing the amount of
acrylamide in thermally
processed foods and permits the production of foods having significantly
reduced levels of
acrylamide. The invention more specifically relates to: a) adding a
combination of two or more
acrylamide-reducing agents when making a fabricated food product and b) the
use of various
acrylamide-reducing agents during the production of potato flakes or other
intermediate products
used in making a fabricated food product.
DESCRIPTION OF RELATED ART:
The chemical acrylamide has long been used in its polymer form in industrial
applications for
water treatment, enhanced oil recovery, papermaking, flocculants, thickeners,
ore processing and
permanent press fabrics. Acrylamide participates as a white crystalline solid,
is odorless, and is
highly soluble in water (2155 g/L at 30 C). Synonyms for acrylamide include 2-
propenamide,
ethylene carboxamide, acrylic acid amide, vinyl amide, and propenoic acid
amide. Acrylamide has a
molecular mass of 71.08, a melting point of 84.5 C, and a boiling point of 125
C at 25 mmHg.
In very recent times, a wide variety of foods have tested positive for the
presence of
acrylamide monomer. Acrylamide has especially been found primarily in
carbohydrate food
products that have been heated or processed at high temperatures. Examples of
foods that have
tested positive for acrylamide include coffee, cereals, cookies, potato chips,
crackers, french-fried
potatoes, breads and rolls, and fried breaded meats. In general, relatively
low contents of acrylamide
have been found in heated protein-rich foods, while relatively high contents
of acrylamide have been
found in carbohydrate-rich foods, compared to non-detectable levels in
unheated and boiled foods.
Reported levels of acrylamide found in various similarly processed foods
include a range of 330 -
2,300 ( g/kg) in potato chips, a range of 300 - 1100 ( g/kg) in French fries,
a range 120 - 180
(gg/kg) in corn chips, and levels ranging from not detectable up to 1400
(gg/kg) in various breakfast
cereals.
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It is presently believed that acrylamide is formed from the presence of amino
acids and
reducing sugars. For example, it is believed that a reaction between free
asparagine, an amino acid
commonly found in raw vegetables, and free reducing sugars accounts for the
majority of acrylamide
found in fried food products. Asparagine accounts for approximately 40% of the
total free amino
acids found in raw potatoes, approximately 18% of the total free amino acids
found in high protein
rye, and approximately 14% of the total free amino acids found in wheat.
The formation of acrylamide from amino acids other than asparagine is
possible, but it has
not yet been confirmed to any degree of certainty. For example, some
acrylamide formation has
been reported from testing glutamine, methionine, cysteine, and aspartic acid
as precursors. These
findings are difficult to confirm, however, due to potential asparagine
impurities in stock amino
acids. Nonetheless, asparagine has been identified as the amino acid precursor
most responsible for
the formation of acrylamide.
Since acrylamide in foods is a recently discovered phenomenon, its exact
mechanism of
formation has not been confirmed. However, it is now believed that the most
likely route for
acrylamide formation involves a Maillard reaction. The Maillard reaction has
long been recognized
in food chemistry as one of the most important chemical reactions in food
processing and can affect
flavor, color, and the nutritional value of the food. The Maillard reaction
requires heat, moisture,
reducing sugars, and amino acids.
The Maillard reaction involves a series of complex reactions with numerous
intermediates,
but can be generally described as involving three steps. The first step of the
Maillard reaction
involves the combination of a free amino group (from free amino acids and/or
proteins) with a
reducing sugar (such as glucose) to form Amadori or Heyns rearrangement
products. The second
step involves 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 -
results in a pool of
flavor intermediates and flavor compounds. The third step of the Maillard
reaction is characterized
by the formation of brown nitrogenous polymers and co-polymers. Using the
Maillard reaction as
the likely route for the formation of acrylamide, Figure 1 illustrates a
simplification of suspected
pathways for the formation of acrylamide starting with asparagine and glucose.
Acrylamide has not been determined to be detrimental to humans, but its
presence in food
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products, especially at elevated levels, is undesirable. As noted previously,
relatively higher
concentrations of acrylamide are found in food products that have been heated
or thermally
processed. The reduction of acrylamide in such food products could be
accomplished by reducing or
eliminating the precursor compounds that form acrylamide, inhibiting the
formation of acrylamide
during the processing of the food, breaking down or reacting the acrylamide
monomer once formed
in the food, or removing acrylamide from the product prior to consumption.
Understandably, each
food product presents unique challenges for accomplishing any of the above
options. For example,
foods that are sliced and cooked as coherent pieces may not be readily mixed
with various additives
without physically destroying the cell structures that give the food products
their unique
characteristics upon cooking. Other processing requirements for specific food
products may
likewise make acrylamide reduction strategies incompatible or extremely
difficult.
By way of example, Figure 2 illustrates well-known prior art methods for
making fried
potato chips from raw potato stock. The raw potatoes, which contain about 80%
or more water by
weight, first proceed to a peeling step 21. After the skins are peeled from
the raw potatoes, the
potatoes are then transported to a slicing step 22. The thickness of each
potato slice at the slicing
step 22 is dependent on the desired the thickness of the final product. An
example in the prior art
involves slicing the potatoes to about 0.053 inches in thickness. These slices
are then transported to
a washing step 23, wherein the surface starch on each slice is removed with
water. The washed
potato slices are then transported to a cooking step 24. This cooking step 24
typically involves
frying the slices in a continuous fryer at, for example, 177 C for
approximately 2.5 minutes. The
cooking step generally reduces the moisture level of the chip to less than 2%
by weight. For
example, a typical fried potato chip exits the fryer at approximately 1.4%
moisture by weight. The
cooked potato chips are then transported to a seasoning step 25, where
seasonings are applied in a
rotation drum. Finally, the seasoned chips proceed to a packaging step 26.
This packaging step 26
usually involves feeding the seasoned chips to one or more weighing devices
that then direct chips to
one or more vertical form, fill, and seal machines for packaging in a flexible
package. Once
packaged, the product goes into distribution and is purchased by a consumer.
Minor adjustments in a number of the potato chip processing steps described
above can result
in significant changes in the characteristics of the final product. For
example, an extended residence
time of the slices in water at the washing step 23 can result in leaching
compounds from the slices
that provide the end product with its potato flavor, color and texture.
Increased residence times or
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heating temperatures at the cooking step 24 can result in an increase in the
Maillard browning levels
in the chip, as well as a lower moisture content. If it is desirable to
incorporate ingredients into the
potato slices prior to frying, it may be necessary to establish mechanisms
that provide for the
absorption of the added ingredients into the interior portions of the slices
without disrupting the
cellular structure of the chip or leaching beneficial compounds from the
slice.
By way of another example of heated food products that represent unique
challenges to
reducing acrylamide levels in the final products, snacks can also be made from
a dough. The term
"fabricated snack" means a snack food that uses as its starting ingredient
something other than the
original and unaltered starchy starting material. For example, fabricated
snacks include fabricated
potato chips that use a dehydrated potato product as a starting material and
corn chips that use masa
flour as its starting material. It is noted here that the dehydrated potato
product can be potato flour,
potato flakes, potato granules, or other forms in which dehydrated potatoes
exist. When any of these
terms are used in this application, it is understood that all of these
variations are included. By way of
example only, and without limitation, examples of "fabricated foods" to which
an acrylamide-
reducing agent can be added include tortilla chips, corn chips, potato chips
made from potato flakes
and/or fresh potato mash, multigrain chips, corn puffs, wheat puffs, rice
puffs, crackers, breads (such
as rye, wheat, oat, potato, white, whole grain, and mixed flours), soft and
hard pretzels, pastries,
cookies, toast, corn tortillas, flour tortillas, pita bread, croissants, pie
crusts, muffins, brownies,
cakes, bagels, doughnuts, cereals, extruded snacks, granola products, flours,
corn meal, masa, potato
flakes, polenta, batter mixes and dough products, refrigerated and frozen
doughs, reconstituted
foods, processed and frozen foods, breading on meats and vegetables, hash
browns, mashed
potatoes, crepes, pancakes, waffles, pizza crust, peanut butter, foods
containing chopped and
processed nuts, jellies, fillings, mashed fruits, mashed vegetables, alcoholic
beverages such as beers
and ales, cocoa, cocoa powder, chocolate, hot chocolate, cheese, animal foods
such as dog and cat
kibble, and any other human or animal food products that are subject to
sheeting or extruding or that
are made from a dough or mixture of ingredients. The use of the term
"fabricated foods" herein
includes fabricated snacks as previously defined. The use of the term "food
products" herein
includes all fabricated snacks and fabricated foods as previously defined.
Referring back to Figure 2, a fabricated potato chip does not require the
peeling step 21, the
slicing step 22, or the washing step 23. Instead, fabricated potato chips
start with, for example,
potato flakes, which are mixed with water and other minor ingredients to form
a dough. This dough
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is then sheeted and cut before proceeding to a cooking step. The cooking step
may involve frying or
baking. The chips then proceed to a seasoning step and a packaging step. The
mixing of the potato
dough generally lends itself to the easy addition of other ingredients, as is
the case with most, if not
all, fabricated foods.
Conversely, the addition of such ingredients to a raw food product, such as
potato slices,
requires that a mechanism be found to allow for the penetration of ingredients
into the cellular
structure of the product. However, the addition of any ingredients in the
mixing step must be done
with the consideration that the ingredients may adversely affect the sheeting,
extruding, or other
processing characteristics of the dough as well as the final chip
characteristics.
It would be desirable to develop one or more methods of reducing the level of
acrylamide in
the end product of heated or thermally processed foods. Ideally, such a
process should substantially
reduce or eliminate the acrylamide in the end product without adversely
affecting the quality and
characteristics of the end product. Further, the method should be easy to
implement and, preferably,
add little or no cost to the overall process.
SUMMARY OF THE INVENTION
The proposed invention involves the reduction of acrylamide in food products.
This
reduction of acrylamide in food is accomplished by exposing the food product
to two or more
acrylamide-reducing agents. For example, the acrylamide-reducing agent
asparaginase, an enzyme
that hydrolyzes asparagine, is used in combination with a free thiol or
divalent or trivalent cations.
Asparaginase can also be used in combination with various amino acids. The
application of the two
or more acrylamide-reducing agents to the food product can be done
simultaneously, in sequence, or
any combination thereof. In the case of fabricated foods, the two acrylamide-
reducing agents can be
mixed with fabricated foods at any point prior to a final heating step. The
above as well as
additional features and advantages of the present invention will become
apparent in the following
written detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
The novel features believed characteristic of the invention are set forth in
the appended
claims. The invention itself, however, as well as a preferred mode of use,
further objectives and
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advantages thereof, will be best understood by reference to the following
detailed description of
illustrative embodiments when read in conjunction with the accompanying
drawings, wherein:
Figure 1 illustrates a simplification of suspected pathways for the formation
of acrylamide
starting with asparagine and glucose.
Figure 2 illustrates well-known prior art methods for making fried potato
chips from raw
potato stock.
Figures 3A and 3B illustrate methods of making a fabricated snack food
according to two
separate embodiments of the invention.
Figure 4 graphically illustrates the acrylamide levels found in a series of
tests in which
cysteine and lysine were added.
Figure 5 graphically illustrates the acrylamide levels found in a series of
tests in which CaC12
was combined with phosphoric acid or citric acid.
Figure 6 graphically illustrates the acrylamide levels found in a series of
tests in which CaCl2
and phosphoric acid were added to potato flakes having various levels of
reducing sugars.
Figure 7 graphically illustrates the acrylamide levels found in a series of
tests in which CaC12
and phosphoric acid were added to potato flakes.
Figure 8 graphically illustrates the acrylamide levels found in a series of
tests in which CaC12
and citric Acid were added to the mix for corn chips.
Figure 9 graphically illustrates the acrylamide levels found in potato chips
fabricated with
cysteine, calcium chloride, and either phosphoric acid or citric acid.
Figure 10 graphically illustrates the acrylamide levels found in potato chips
when calcium
chloride and phosphoric acid are added at either the flakes making step or the
chip fabrication step.
Figure 11 graphically illustrates the effect of asparaginase and buffering on
acrylamide level
in potato chips.
Figure 12 graphically illustrates the acrylamide levels found in potato chips
fried in oil
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containing rosemary.
Figure 13 graphically illustrates the effect of the addition of an oxidizing
agent or reducing
agent to an acrylamide-reducing agent having a free thiol.
Figure 14 graphically illustrates the effect on acrylamide levels of
polyvalent cations which
lower pH.
Figure 15 graphically illustrates the effect on pH of calcium chloride or
sodium chloride to a
0.5 M phosphate and a 0.5 M acetate buffer.
DETAILED DESCRIPTION
The formation of acrylamide in thermally processed foods requires a source of
carbon and a
source of nitrogen. It is hypothesized that carbon is provided by a
carbohydrate source and nitrogen
is provided by a protein source or amino acid source. Many plant-derived food
ingredients such as
rice, wheat, corn, barley, soy, potato and oats contain asparagine and are
primarily carbohydrates
having minor amino acid components. Typically, such food ingredients have a
small amino acid
pool, which contains other amino acids in addition to asparagine.
By "thermally processed" is meant food or food ingredients wherein components
of the food,
such as a mixture of food ingredients, are heated at temperatures of at least
80 C. Preferably the
thermal processing of the food or food ingredients takes place at temperatures
between about 100 C
and 205 C. The food ingredient may be separately processed at elevated
temperature prior to the
formation of the final food product. An example of a thermally processed food
ingredient is potato
flakes, which is formed from raw potatoes in a process that exposes the potato
to temperatures as
high as 170 C. (The terms "potato flakes", "potato granules", and "potato
flour" are used
interchangeably herein, and are meant to denote any potato based, dehydrated
product.) Examples
of other thermally processed food ingredients include processed oats, par-
boiled and dried rice,
cooked soy products, corn masa, roasted coffee beans and roasted cacao beans.
Alternatively, raw
food ingredients can be used in the preparation of the final food product
wherein the production of
the final food product includes a thermal heating step. One example of raw
material processing
wherein the final food product results from a thermal heating step is the
manufacture of potato chips
from raw potato slices by the step of frying at a temperature of from about
100 C to about 205 C or
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the production of french fries fried at similar temperatures. As referred to
herein, the thermally-
processed foods include, by way of example and without limitation, all of the
foods previously listed
as examples of fabricated snacks and fabricated foods, as well as french
fries, yam fries, other tuber
or root materials, cooked vegetables including cooked asparagus, onions, and
tomatoes, coffee
beans, cocoa beans, cooked meats, dehydrated fruits and vegetables, heat-
processed animal feed,
tobacco, tea, roasted or cooked nuts, soybeans, molasses, sauces such as
barbecue sauce, plantain
chips, apple chips, fried bananas, and other cooked fruits.
In accordance with the present invention, however, a significant formation of
acrylamide has
been found to occur when the amino acid asparagine is heated in the presence
of a reducing sugar.
Heating other amino acids such as lysine and alanine in the presence of a
reducing sugar such as
glucose does not lead to the formation of acrylamide. But, surprisingly, the
addition of other amino
acids to the asparagine-sugar mixture can increase or decrease the amount of
acrylamide formed.
Having established the rapid formation of acrylamide when asparagine is heated
in the
presence of a reducing sugar, a reduction of acrylamide in thermally processed
foods can be
achieved by inactivating the asparagine. By "inactivating" is meant removing
asparagine from the
food or rendering asparagine non-reactive along the acrylamide formation route
by means of
conversion or binding to another chemical that interferes with the formation
of acrylamide from
asparagine.
1. EFFECT OF CYSTEINE, LYSINE, GLUTAMINE AND GLYCINE ON ACRYLAMIDE FORMATION
Since asparagine reacts with glucose to form acrylamide, increasing the
concentration of
other free amino acids may affect the reaction between asparagine with glucose
and reduce
acrylamide formation. For this experiment, a solution of asparagine (0.176 %)
and glucose (0.4%)
was prepared in pH 7.0 sodium phosphate buffer. Four other amino acids,
glycine (GLY), lysine
(LYS), glutamine (GLN), and cysteine (CYS) were added at the same
concentration as glucose on a
molar basis. The experimental design was full factorial without replication so
all possible
combinations of added amino acids were tested. The solutions were heated at
120 C for 40 minutes
before measuring acrylamide. Table 1 below shows the concentrations and the
results.
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Glucose ASN GLY LYS GLN CYS Acrylamide
Order % % % % % % ppb
1 0.4 0.176 0 0 0 0 1679
2 0.4 0.176 0 0 0 0.269 4
3 0.4 0.176 0 0 0.324 0 5378
4 0.4 0.176 0 0 0.324 0.269 7
0.4 0.176 0 0.325 0 0 170
6 0.4 0.176 0 0.325 0 0.269 7
7 0.4 0.176 0 0.325 0.324 0 1517
8 0.4 0.176 0 0.325 0.324 0.269 7
9 0.4 0.176 0.167 0 0 0 213
0.4 0.176 0.167 0 0 0.269 6
11 0.4 0.176 0.167 0 0.324 0 2033
12 0.4 0.176 0.167 0 0.324 0.269 4
13 0.4 0.176 0.167 0.325 0 0 161
14 0.4 0.176 0.167 0.325 0 0.269 4
0.4 0.176 0.167 0.325 0.324 0 127
16 0.4 0.176 0.167 0.325 0.324 0.269 26
TABLE 1: Effect of Cysteine, Lysine, Glutamine and Glycine on Acrylamide
Formation
As shown in the table above, glucose and asparagine without any other amino
acid formed
1679 ppb acrylamide. The added amino acids had three types of effects.
1) Cysteine almost eliminated acrylamide formation. All treatments with
cysteine had
5 less than 25 ppb acrylamide (a 98% reduction).
2) Lysine and glycine reduced acrylamide formation but not as much as
cysteine. All
treatments with lysine and/or glycine but without glutamine and cysteine had
less than 220 ppb
acrylamide (a 85% reduction).
3) Surprisingly, glutamine increased acrylamide formation to 5378 ppb (200%
increase).
10 Glutamine plus cysteine did not form acrylamide. Addition of glycine and
lysine to glutamine
reduced acrylamide formation.
These tests demonstrate the effectiveness of cysteine, lysine, and glycine in
reducing
acrylamide formation. However, the glutamine results demonstrate that not all
amino acids are
effective at reducing acrylamide formation. The combination of cysteine,
lysine, or glycine with an
15 amino acid that alone can accelerate the formation of acrylamide (such as
glutamine) can likewise
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reduce the acrylamide formation.
II. EFFECT OF CYSTEINE, LYSINE, GLUTAMINE, AND METHIONINE AT DIFFERENT
CONCENTRATIONS AND TEMPERATURES
As reported above, cysteine and lysine reduced acrylamide when added at the
same
concentration as glucose. A follow up experiment was designed to answer the
following questions:
1) How do lower concentrations of cysteine, lysine, glutamine, and methionine
effect
acrylamide formation?
2) Are the effects of added cysteine and lysine the same when the solution is
heated at
120 C and 150 C?
A solution of asparagine (0.176 %) and glucose (0.4%) was prepared in pH 7.0
sodium
phosphate buffer. Two concentrations of amino acid (cysteine (CYS), lysine
(LYS), glutamine
(GLN), or methionine (MET)) were added. The two concentrations were 0.2 and
1.0 moles of amino
acid per mole of glucose. In half of the tests, two ml of the solutions were
heated at 120 C for 40
minutes; in the other half, two ml were heated at 150 C for 15 minutes. After
heating, acrylamide
was measured by GC-MS, with the results shown in Table 2. The control was
asparagine and
glucose solution without an added amino acid.
Acrylamide level
Amino acid/ Control Amino Acid Percentage Amino Acid Percentage
Temperature @ Conc. 0.2 Of Control @ Conc. 1.0 Of Control
LYS-120 C 1332 ppb 1109 ppb 83% 280 b 21%
CYS-120 C 1332 ppb 316 ppb 24% 34 b 3%
LYS-150 C 3127 ppb 1683 b 54% 536 b 17%
CYS-150 C 3127 ppb 1146 ppb 37% 351 b 11%
GLN-120 C 1953 ppb 4126 ppb 211% 6795 b 348%
MET-120 C 1953 ppb 1978 b 101% 1132 b 58%
GLN-150 C 3866 ppb 7223 ppb 187% 9516 pb 246%
MET-150 C 3866 ppb 3885 ppb 100% 3024 b 78%
TABLE 2: Effect of Temperature and Concentration of Amino Acids on Acrylamide
Level
In the tests with cysteine and lysine, a control formed 1332 ppb of acrylamide
after 40
minutes at 120 C, and 3127 ppb of acrylamide after 15 minutes at 150 C.
Cysteine and lysine
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reduced acrylamide formation at 120 C and 150 C, with the acrylamide reduction
being roughly
proportional to the concentration of added cysteine or lysine.
In the tests with glutamine and methionine, a control formed 1953 ppb of
acrylamide after 40
minutes at 120 C and a control formed 3866 ppb of acrylamide after 15 minutes
at 150 C.
Glutamine increased acrylamide formation at 120 C and 150 C. Methionine at 0.2
mole/mole of
glucose did not affect acrylamide formation. Methionine at 1.0 mole/mole of
glucose reduced
acrylamide formation by less than fifty percent.
111. EFFECT OF NINETEEN AMINO ACIDS ON ACRYLAMIDE FORMATION IN GLUCOSE AND
ASPARAGINE SOLUTION
The effect of four amino acids (lysine, cysteine, methionine, and glutamine)
on acrylamide
formation was described above. Fifteen additional amino acids were tested. A
solution of
asparagine (0.176 %) and glucose (0.4%) was prepared in pH 7.0 sodium
phosphate buffer. The
fifteen amino acids were added at the same concentration as glucose on a molar
basis. The control
contained asparagine and glucose solution without any other amino acid. The
solutions were heated
at 120 C for 40 minutes before measuring acrylamide by GC-MS. The results are
given in Table 3
below.
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Acr lamide Formed
Amino Acid ppb % of Control
Control 959 100
Histidine 215 22
Alanine 478 50
Methionine 517 54
Glutamic Acid 517 54
Aspartic Acid 529 55
Proline 647 67
Phenylalanine 648 68
Valine 691 72
Arginine 752 78
Tryptophan 1059 111
Threonine 1064 111
Tyrosine 1091 114
Leucine 1256 131
Serine 1296 135
Isoleucine 1441 150
TABLE 3: Effect of Other Amino Acids on Acrylamide Formation
As seen in the table above, none of the fifteen additional amino acids were as
effective as
cysteine, lysine, or glycine in reducing acrylamide formation. Nine of the
additional amino acids
reduced acrylamide to a level between 22-78% of control, while six amino acids
increased
acrylamide to a level between 111-150 % of control.
Table 4 below summarizes the results for all amino acids, listing the amino
acids in the order
of their effectiveness. Cysteine, lysine, and glycine were effective
inhibitors, with the amount of
acrylamide formed less than 15% of that formed in the control. The next nine
amino acids were less
effective inhibitors, having a total acrylamide formation between 22-78% of
that formed in the
control. The next seven amino acids increased acrylamide. Glutamine caused the
largest increase of
acrylamide, showing 320% of control.
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Acrylamide produced
Amino Acid as % of Control
Control 100%
Cysteine 0%
Lysine 10 %
Glycine 13 %
Histidine 22 %
Alanine 50 %
Methionine 54 %
Glutamic Acid 54 %
Aspartic Acid 55 %
Proline 67 %
Phenylalanine 68 %
Valine 72 %
Arginine 78 %
Tryptophan 111 %
Threonine 111 %
Tyrosine 114 %
Leucine 131 %
Serine 135%
Isoleucine 150 %
Glutamine 320 %
Table 4: Acrylamide Formation in the Presence of 19 Amino Acids
IV. POTATO FLAKES WITH 750 PPM OF ADDED L-CYSTEINE
Test potato flakes were manufactured with 750 ppm (parts per million) of added
L-cysteine.
The control potato flakes did not contain added L-cysteine. Three grams of
potato flakes were
weighed into a glass vial. After tightly capping, the vials were heated for 15
minutes or 40 minutes
at 120 C. Acrylamide was measured by GC-MS in parts per billion (ppb).
Potato Flakes Acrylamide (ppb) Acrylamide Acrylamide (ppb) Acrylamide
Min at 120 C Reduction 15 Min 40 Min at 120 C Reduction 40 Min
Control 1662 -- 9465 --
750 ppm 653 60% 7529 20%
Cysteine
TABLE 5: Reduction of Acrylamide over Time with Cysteine
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V. BAKED FABRICATED POTATO CHIPS
Given the above results, preferred embodiments of the invention have been
developed in
which cysteine or lysine was added to the formula for a fabricated snack food,
in this case baked,
fabricated potato chips. The process for making this product is shown in
Figure 3A. In a dough
preparation step 30, potato flakes, water, and other ingredients are combined
to form a dough. (The
terms "potato flakes," "potato granules," and "potato flour" are used
interchangeably herein and all
are intended to encompass all dried flake or powder preparations, regardless
of particle size.) In a
sheeting step 31, the dough is run through a sheeter, which flattens the
dough, and is then cut into
discrete pieces. In a cooking step 32, the cut pieces are baked until they
reach a specified color and
water content. The resulting chips are then seasoned in a seasoning step 33
and placed in packages in
a packaging step 34.
A first embodiment of the invention is demonstrated by use of the process
described above.
To illustrate this embodiment, a comparison is made between a control and test
batches to which
were added either one of three concentrations of cysteine or one concentration
of lysine.
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Ingredient Control Cysteine Cysteine Cysteine Lysine
#1 #2 #3
Potato flakes & modified starch (g) 5496 5496 5496 5496 5496
Sugar (g) 300 300 300 300 300
Oil (g) 90 90 90 90 90
Leavening agents (g) 54 54 54 54 54
Emulsifier (g) 60 60 60 60 60
L-Cysteine (dissolved in water)' (g) 0 1.8 4.2 8.4 0
L-Lysine monohydrochloride (g) 0 0 0 0 42
Total Dry (g) 6000 6001.8 6004.2 6008.4 6042
Water (ml) 3947 3947 3947 3947 3947
Measurements after Cooking Chips
H20, % 2.21 1.73 % 2.28% 2.57% 2.68%
Oil, % 1.99 2.15 % 2.05 % 2.12 % 1.94 %
Acrylamide (ppb) 1030 620 166 104 456
Color L 72.34 76.53 79.02 78.36 73.2
A 1.99 -1.14 -2.02 -2.14 1.94
B 20.31 25.52 23.2 23.0 25.77
TABLE 6: Effect of Lysine and Various Levels of Cysteine on Acrylamide Level
In all batches, the dry ingredients were first mixed together, then oil was
added to each dry
blend and mixed. The cysteine or lysine was dissolved in the water prior to
adding to the dough. The
moisture level of the dough prior to sheeting was 40% to 45% by weight. The
dough was sheeted to
produce a thickness of between 0.020 and 0.030 inches, cut into chip-sized
pieces, and baked.
After cooking, testing was performed for moisture, oil, and color according to
the Hunter L-
A-B scale. Samples were tested to obtain acrylamide levels in the finished
product. Table 6 above
shows the results of these analyses.
In the control chips, the acrylamide level after final cooking was 1030 ppb.
Both the addition
of cysteine, at all the levels tested, and lysine reduced the final acrylamide
level significantly. Figure
4 shows the resulting acrylamide levels in graphical form. In this drawing,
the level of acrylamide
detected in each sample is shown by a shaded bar 402. Each bar has a label
listing the appropriate
test immediately below and is calibrated to the scale for acrylamide on the
left of the drawing. Also
shown for each test is the moisture level of the chip produced, seen as a
single point 404. The values
1 It is expected that the D- isomer or a racemic mixture of both the D- and L-
isomers of the amino acids would be
equally effective, although the L- isomer is likely to be the best and least
expensive source.
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CA 02618225 2008-01-18
for these points 404 are calibrated to the scale for percentage of moisture
shown on the right of the
drawing. A line 406 connects the individual points 404 for greater visibility.
Because of the marked
effect of lower moisture on the level of acrylamide, it is important to note a
moisture level in order to
properly evaluate the activity of any acrylamide-reducing agents. As used
herein, an acrylamide-
reducing agent is an additive that reduces acylamide content in the final
product of a thermally-
processed food as compared to the same final product in which the agent was
not added.
Adding cysteine or lysine to the dough significantly lowers the level of
acrylamide present in
the finished product. The cysteine samples show that the level of acrylamide
is lowered in roughly a
direct proportion to the amount of cysteine added. Consideration must be made,
however, for the
collateral effects on the characteristics (such as color, taste, and texture)
of the final product from the
addition of an amino acid to the manufacturing process.
Additional tests were also run, using added cysteine, lysine, and combinations
of each of the
two amino acids with CaC12. These tests used the same procedure as described
in the tests above, but
used potato flakes having varying levels of reducing sugars and varying
amounts of amino acids and
CaC12 added. In Table 7 below, lot 1 of potato flakes had 0.81 % reducing
sugars (this portion of the
table reproduces the results from the test shown above), lot 2 had 1.0% and
lot 3 had 1.8% reducing
sugars.
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Reducing CaC12 Cysteine Lysine % Finish Finish Acrylamide
Sugar % Wt % of ppm of of total H2O wt color ppb
total dry total dry dry- % value
0.81 0 0 0 2.21 72.34 1030
0.81 0 300 0 1.73 76.53 620
0.81 0 700 0 2.28 79.02 166
0.81 0 1398 0 2.57 78.36 104
0.81 0 0 0.685 2.68 73.20 456
1.0 0 0 0 1.71 72.68 599
1.0 0 0 0 1.63 74.44 1880
1.0 0 0 0 1.69 71.26 1640
1.0 0 0 0 1.99 71.37 1020
1.0 0 700 0 2.05 75.81 317
1.0 0.646 0 0.685 1.74 73.99 179
1.8 0 0 0 1.80 73.35 464
1.8 0 0 0 1.61 72.12 1060
1.8 0 700 0 1.99 75.27 290
1.8 0 1398 0 1.96 75.87 188
1.8 0 0 0.685 1.90 76.17 105
1.8 0.646 0 0.685 2.14 75.87 47
1.8 0.646 700 0 1.83 77.23 148
TABLE 7: Effect of Varying Concentration of Cysteine, Lysine, Reducing Sugars
As shown by the data in this table, the addition of either cysteine or lysine
provides
significant improvement in the level of acrylamide at each level of reducing
sugars tested. The
combination of lysine with calcium chloride provided an almost total
elimination of acrylamide
produced, despite the fact that this test was run with the highest level of
reducing sugars.
VI. TESTS IN SLICED, FRIED POTATO CHIPS
A similar result can be achieved with potato chips made from potato slices.
However, the
desired amino acid cannot be simply mixed with the potato slices, as with the
embodiments
illustrated above, since this would destroy the integrity of the slices. In
one embodiment, the potato
slices are immersed in an aqueous solution containing the desired amino acid
additive for a period of
time sufficient to allow the amino acid to migrate into the cellular structure
of the potato slices. This
can be done, for example, during the washing step 23 illustrated in Figure 2.
Table 8 below shows the result of adding one weight percent of cysteine to the
wash
treatment that was described in step 23 of Figure 2 above. All washes were at
room temperature for
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CA 02618225 2008-01-18
the time indicated; the control treatments had nothing added to the water. The
chips were fried in
cottonseed oil at 178 C for the indicated time.
Fry Time Finished Finished Finished
(seconds) H2O wt % oil wt % Acrylamide
Control - 2-3 min wash 140 1.32% 42.75% 323 ppb
1 % cysteine - 15 min wash 140 .86 % 45.02 % 239 ppb
trol - 2-3 min wash 110 1.72 % 40.87 % 278 ppb
Control - 15 min wash 110 1.68 % 41.02 % 231 ppb
1 % Cysteine - 15 min wash 110 1.41 % 44.02 % 67 ppb
TABLE 8: Effect of Cysteine in Wash Water of Potato Slices on Acrylamide
As shown in this table, immersing potato slices of .053 inch thickness for 15
minutes in an
aqueous solution containing a concentration of one weight percent of cysteine
is sufficient to reduce
the acrylamide level of the final product on the order of 100-200 ppb.
The invention has also been demonstrated by adding cysteine to the corn dough
(or masa) for
tortilla chips. Dissolved L-cysteine was added to cooked corn during the
milling process so that
cysteine was uniformly distributed in the masa produced during milling. The
addition of 600 ppm of
L-cysteine reduced acrylamide from 190 ppb in the control product to 75 ppb in
the L-cysteine
treated product.
Any number of amino acids can be used with the invention disclosed herein, as
long as
adjustments are made for the collateral effects of the additional
ingredient(s), such as changes to the
color, taste, and texture of the food. Although all examples shown utilize a-
amino acids (where the -
NH2 group is attached to the alpha carbon atom), the applicants anticipate
that other isomers, such as
(3- or y-amino acids can also be used, although 0- and y-amino acids are not
commonly used as food
additives. The preferred embodiment of this invention uses cysteine, lysine,
and/or glycine.
However, other amino acids, such as histidine, alanine, methionine, glutamic
acid, aspartic acid,
proline, phenylalanine, valine, and arginine may also be used. Such amino
acids, and in particular
cysteine, lysine, and glycine, are relatively inexpensive and commonly used as
food additives in
certain foods. These preferred amino acids can be used alone or in combination
in order to reduce
the amount of acrylamide in the final food product. Further, the amino acid
can be added to a food
product prior to heating by way of either adding the commercially available
amino acid to the
starting material of the food product or adding another food ingredient that
contains a high
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CA 02618225 2008-01-18
concentration level of the free amino acid. For example, casein contains free
lysine and gelatin
contains free glycine. Thus, when Applicants indicate that an amino acid is
added to a food
formulation, it will be understood that the amino acid may be added as a
commercially available
amino acid or as a food having a concentration of the free amino acid(s) that
is greater than the
naturally occurring level of asparagine in the food.
The amount of amino acid that should be added to the food in order to reduce
the acrylamide
levels to an acceptable level can be expressed in several ways. In order to be
commercially
acceptable, the amount of amino acid added should be enough to reduce the
final level of acrylamide
production by at least twenty percent (20%) as compared to a product that is
not so treated. More
preferably, the level of acrylamide production should be reduced by an amount
in the range of thirty-
five to ninety-five percent (35-95%). Even more preferably, the level of
acrylamide production
should be reduced by an amount in the range of fifty to ninety-five percent
(50-95%). In a preferred
embodiment using cysteine, it has been determined that the addition of at
least 100 ppm can be
effective in reducing acrylamide. However, a preferred range of cysteine
addition is between 100
ppm and 10,000 ppm, with the most preferred range in the amount of about 1,000
ppm. In preferred
embodiments using other effective amino acids, such as lysine and glycine, a
mole ratio of the added
amino acid to the reducing sugar present in the product of at least 0.1 mole
of amino acid to one
mole of reducing sugars (0.1:1) has been found to be effective in reducing
acrylamide formation.
More preferably the molar ratio of added amino acid to reducing sugars should
be between 0.1:1 and
2:1, with a most preferable ratio of about 1:1.
The mechanisms by which the select amino acids reduce the amount of acrylamide
found are
not presently known. Possible mechanisms include competition for reactant and
dilution of the
precursor, which will create less acrylamide, and a reaction mechanism with
acrylamide to break it
down." Possible mechanisms include (1) inhibition of Maillard reaction, (2)
consumption of glucose
and other reducing sugars, and (3) reaction with acrylamide. Cysteine, with a
free thiol group, acts as
an inhibitor of the Maillard reaction. Since acrylamide is believed to be
formed from asparagine by
the Maillard reaction, cysteine should reduce the rate of the Maillard
reaction and acrylamide
formation. Lysine and glycine react rapidly with glucose and other reducing
sugars. If glucose is
consumed by lysine and glycine, there will be less glucose to react with
asparagine to form
acrylamide. The amino group of amino acids can react with the double bond of
acrylamide, a
Michael addition. The free thiol of cysteine can also react with the double
bond of acrylamide.
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CA 02618225 2008-01-18
It should be understood that adverse changes in the characteristics of the
final product, such
as changes in color, taste, and texture, could be caused by the addition of an
amino acid. These
changes in the characteristics of the product in accordance with this
invention can be compensated
by various other means. For example, color characteristics in potato chips can
be adjusted by
controlling the amount of sugars in the starting product. Some flavor
characteristics can be changed
by the addition of various flavoring agents to the end product. The physical
texture of the product
can be adjusted by, for example, the addition of leavening agents or various
emulsifiers.
VII. EFFECT OF DI- AND TRIVALENT CATIONS ON ACRYLAMIDE FORMATION
Another embodiment of the invention involves reducing the production of
acrylamide by the
addition of a divalent or trivalent cation to a formula for a snack food prior
to the cooking or thermal
processing of that snack food. Chemists will understand that cations do not
exist in isolation, but are
found in the presence of an anion having the same valence. Although reference
is made herein to the
salt containing the divalent or trivalent cation, it is the cation present in
the salt that is believed to
provide a reduction in acrylamide formation by reducing the solubility of
asparagine in water. These
cations are also referred to herein as a cation with a valence of at least
two. Interestingly, cations of
a single valence are not effective in use with the present invention. In
choosing an appropriate
compound containing the cation having a valence of at least two in combination
with an anion, the
relevant factors are water solubility, food safety, and least alteration to
the characteristics of the
particular food. Combinations of various salts can be used, even though they
are discussed herein
only as individuals salts.
Chemists speak of the valence of an atom as a measure of its ability to
combine with other
elements. Specifically, a divalent atom has the ability to form two ionic
bonds with other atoms,
while a trivalent atom can form three ionic bonds with other atoms. A cation
is a positively charged
ion, that is, an atom that has lost one or more electrons, giving it a
positive charge. A divalent or
trivalent cation, then, is a positively charged ion that has availability for
two or three ionic bonds,
respectively.
Simple model systems can be used to test the effects of divalent or trivalent
cations on
acrylamide formation. Heating asparagine and glucose in 1:1 mole proportions
can generate
acrylamide. Quantitative comparisons of acrylamide content with and without an
added salt
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CA 02618225 2008-01-18
measures the ability of the salt to promote or inhibit acrylamide formation.
Two sample preparation
and heating methods were used. One method involved mixing the dry components,
adding an equal
amount of water, and heating in a loosely capped vial. Reagents concentrated
during heating as most
of the water escaped, duplicating cooking conditions. Thick syrups or tars can
be produced,
complicating recovery of acrylamide. These tests are shown in Examples 1 and 2
below.
A second method using pressure vessels allowed more controlled experiments.
Solutions of
the test components were combined and heated under pressure. The test
components can be added at
the concentrations found in foods, and buffers can duplicate the pH of common
foods. In these tests,
no water escapes, simplifying recovery of acrylamide, as shown in Example 3
below.
VIII. DIVALENT, TRIVALENT CATIONS DECREASE ACRYLAMIDE, MONOVALENT DON'T
As Example 1, a 20 mL (milliliter) glass vial containing L-asparagine
monohydrate (0.15 g,1
mmole), glucose (0.2 g, 1 mmole) and water (0.4 mL) was covered with aluminum
foil and heated in
a gas chromatography (GC) oven programmed to heat from 40 to 220 C at 20
/minute, hold two
minutes at 220 C, and cool from 220 to 40 C at 20 /min. The residue was
extracted with water and
analyzed for acrylamide using gas chromatography-mass spectroscopy (GC-MS).
Analysis found
approximately 10,000 ppb (parts/billion) acrylamide. Two additional vials
containing L-asparagine
monohydrate (0.13 g, 1 mmole), glucose (0.2 g, 1 mmole), anhydrous calcium
chloride (0.1 g, 1
mmole) and water (0.4 mL) were heated and analyzed. Analysis found 7 and 30
ppb acrylamide, a
greater than ninety-nine percent reduction.
Given the surprising result that calcium salts strongly reduced acrylamide
formation, further
screening of salts was performed and identified divalent and trivalent cations
(magnesium,
aluminum) as producing a similar effect. It is noted that similar experiments
with monovalent
cations, i.e. 0.1/0.2 g sodium bicarbonate and ammonium carbonate (as ammonium
carbamate and
ammonium bicarbonate) increased acrylamide formation, as seen in Table 9
below.
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Salt Micro Mole Micromole Acrylamide
Salt after heating, ppb
None (control) 0 9857
Sodium bicarbonate 1200 13419
Ammonium carbonate 1250 22027
Ammonium carbonate 2500 47897
TABLE 9
IX. CALCIUM CHLORIDE AND MAGNESIUM CHLORIDE
As Example 2, a similar test to that described above was performed, but
instead of using
anhydrous calcium chloride, two different dilutions of each of calcium
chloride and magnesium
chloride were used. Vials containing L-asparagine monohydrate (0.15 g,1 mmole)
and glucose (0.2
g, 1 mmole) were mixed with one of the following:
0.5 mL water (control),
0.5 mL 10% calcium chloride solution (0.5 mmole),
0.05 mL 10% calcium chloride solution (0.05 mmole) plus 0.45 mL water,
0.5 mL 10% magnesium chloride solution (0.5 mmole), or
0.05 mL 10% magnesium chloride solution (0.05 mmole) plus 0.45 mL water.
Duplicate samples were heated and analyzed as described in Example 1. Results
were
averaged and summarized in Table 10 below:
Salt ID Amt added Acrylamide formed Acrylamide
Micromoles Micromoles reduction
None (control) 0 408 0
Calcium chloride 450 293 27%
Calcium chloride 45 864 None
Magnesium chloride 495 191 53%
Magnesium chloride 50 2225 None
TABLE 10: Effect of Calcium Chloride, Magnesium Chloride on Acrylamide
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X. PH AND BUFFERING EFFECTS
As mentioned above, this test, Example 3, did not involve the loss of water
from the
container, but was done under pressure. Vials containing 2 mL of buffered
stock solution (15 mM
asparagine, 15 mM glucose, 500 mM phosphate or acetate) and 0.1 mL salt
solution (1000 mM)
were heated in a Parr bomb placed in a gas chromatography oven programmed to
heat from 40 to
150 C at 20 /minute and hold at 150 C for 2 minutes. The bomb was removed from
the oven and
cooled for 10 minutes. The contents were extracted with water and analyzed for
acrylamide
following the GC-MS method. For each combination of pH and buffer, a control
was run without an
added salt, as well as with the three different salts. Results of duplicate
tests were averaged and
summarized in Table 11 below:
Salt with Divalent or pH Buffer Mcg Acrylamide Acrylamide
Trivalent Cation Used Salt added Control Reduction
Calcium chloride 5.5 Acetate 337 550 19%
Calcium chloride 7.0 Acetate 990 1205 18%
Calcium chloride 5.5 Phosphate 154 300 49%
Calcium chloride 7.0 Phosphate 762 855 11%
Magnesium chloride 5.5 Acetate 380 550 16%
Magnesium chloride 7.0 Acetate 830 1205 31%
Magnesium chloride 5.5 Phosphate 198 300 34%
Magnesium chloride 7.0 Phosphate 773 855 10%
Potassium aluminum 5.5 Acetate 205 550 31%
sulfate
Potassium aluminum 7.0 Acetate 453 1205 62%
sulfate
Potassium aluminum 5.5 Phosphate 64 300 79%
sulfate
Potassium aluminum 7.0 Phosphate 787 855 8%
sulfate
TABLE 11: Effect of PH and Buffer on Divalent/Trivalent Cations Reduction of
Acrylamide
Across the three salts used, the greatest reductions occurred in pH 7 acetate
and pH 5.5
phosphate. Only small reductions were found in pH 5.5 acetate and pH 7
phosphate.
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XI. RAISING CALCIUM CHLORIDE LOWERS ACRYLAMIDE
Following the model systems results, a small-scale laboratory test was run in
which calcium
chloride was added to potato flakes before heating. Three ml of a 0.4%, 2%, or
10% calcium
chloride solution was added to 3 g of potato flakes. The control was 3 g of
potato flakes mixed with
3 ml of de-ionized water. The flakes were mixed to form a relatively uniform
paste and then heated
in a sealed glass vial at 120 C for 40 min. Acrylamide after heating was
measured by GC-MS.
Before heating, the control potato flakes contained 46 ppb of acrylamide.
Test results are reflected in Table 12 below.
Mixture ID Acrylamide, ppb Acrylamide Reduction
Control (water) 2604 None
CaC12 0.4% solution 1877 28%
CaC12 2% solution 338 76%
CaC12 10% solution 86 97%
TABLE 12: Effect of Calcium Chloride Solution Strength on Acrylamide Reduction
Given the results from above, tests were conducted in which a calcium salt was
added to the
formula for a fabricated snack food, in this case baked fabricated potato
chips. The process for
making baked fabricated potato chips consists of the steps shown in Figure 3B.
The dough
preparation step 35 combines potato flakes with water, the cation/anion pair
(which in this case is
calcium chloride) and other minor ingredients, which are thoroughly mixed to
form a dough. (Again,
the term "potato flakes" is intended herein to encompass all dried potato
flake, granule, or powder
preparations, regardless of particle size.) In the sheeting/cutting step 36,
the dough is run through a
sheeter, which flattens the dough, and then is cut into individual pieces. In
the cooking step 37, the
formed pieces are cooked to a specified color and water content. The resultant
chips are then
seasoned in seasoning step 38 and packaged in packaging step 39.
In a first test, two batches of fabricated potato chips were prepared and
cooked according to
the recipe given in Table 13; with the only difference between the batches was
that the test batch
contained calcium chloride. In both batches, the dry ingredients were first
mixed together, then oil
was added to each dry blend and mixed. The calcium chloride was dissolved in
the water prior to
adding to the dough. The moisture level of the dough prior to sheeting was 40%
to 45% by weight.
The dough was sheeted to produce a thickness of between 0.020 and 0.030
inches, cut into chip-
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CA 02618225 2008-01-18
sized pieces, and baked.
After cooking, testing was performed for moisture, oil, and color according to
the Hunter L-
ab scale. Samples were tested to obtain acrylamide levels in the finished
product. Table 13 below
also shows the results of these analyses.
Ingredient Control CaC12 Test
Potato flakes and modified starch (g) 5496 5496
Sugar (g) 300 300
Oil (g) 90 90
Leavening agents (g) 54 54
Emulsifier (g) 60 60
Calcium Chloride (dissolved in water) (g) 0 39
Total Dry Mix (g) 6000 6039
Water (ml) 3947 3947
Tests Performed after Chips Cooked
H2O, % 2.21 2.58
Oil, % 1.99 2.08
Acrylamide, ppb 1030 160
L 72.34 76.67
A 1.99 -.67
B 20.31 24.21
TABLE 13: Effect of CaCl2 on Acrylamide in Chips
As these results show, the addition of calcium chloride to the dough in a
ratio by weight of
calcium chloride to potato flakes of roughly 1 to 125 significantly lowers the
level of acrylamide
present in the finished product, lowering the final acrylamide levels from
1030 ppb to 160 ppb.
Additionally, the percentages of oil and water in the final product do not
appear to have been
affected by the addition of calcium chloride. It is noted, however, that CaC12
can cause changes in
the taste, texture, and color of the product, depending on the amount used.
The level of divalent or trivalent cation that is added to a food for the
reduction of acrylamide
can be expressed in a number of ways. In order to be commercially acceptable,
the amount of cation
added should be enough to reduce the final level of acrylamide production by
at least twenty percent
(20%). More preferably, the level of acrylamide production should be reduced
by an amount in the
range of thirty-five to ninety-five percent (35-95%). Even more preferably,
the level of acrylamide
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CA 02618225 2008-01-18
production should be reduced by an amount in the range of fifty to ninety-five
percent (50-95%). To
express this in a different manner, the amount of divalent or trivalent cation
to be added can be given
as a ratio between the moles of cation to the moles of free asparagine present
in the food product.
The ratio of the moles of divalent or trivalent cation to moles of free
asparagine should be at least
one to five (1:5). More preferably, the ratio is at least one to three (1:3),
and more preferably still,
one to two (1:2). In the presently preferred embodiment, the ratio of moles of
cations to moles of
asparagine is between about 1:2 and 1:1. In the case of magnesium, which has
less effect on the
product taste than calcium, the molar ratio of cation to asparagine can be as
high as about two to one
(2:1).
Additional tests were run, using the same procedure as described above, but
with different
lots of potato flakes containing different levels of reducing sugars and
varying amounts of calcium
chloride added. In Table 14 below, the chips having 0.8 % reducing sugars
reproduce the test shown
above.
CaC12 Reducing Moisture Color L Acrylamide
(g) Sugar % % Value ppb
0 0.8 2.21 72.34 1030
39 0.8 2.58 76.67 160
0 1.0 1.80 73.35 464
0 1.0 1.61 72.12 1060
17.5 1.0 1.82 74.63 350
39 1.0 2.05 76.95 80
39 1.0 1.98 75.86 192
0 1.8 1.99 71.37 1020
0 1.8 1.71 72.68 599
0 1.8 1.69 71.26 1640
0 1.8 1.63 74.44 1880
39 1.8 1.89 76.59 148
39 1.8 1.82 75.14 275
TABLE 14: Effect of CaC12 Across Varying Levels of Reducing Sugars & Cation
Levels
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As seen in this table, the addition of CaC12 consistently reduces the level of
acrylamide in the
final product, even when the weight ratio of added CaC12 to potato flakes is
lower than 1:250.
Any number of salts that form a divalent or trivalent cation (or said another
way, produce a
cation with a valence of at least two) can be used with the invention
disclosed herein, as long as
adjustments are made for the collateral effects of this additional ingredient.
The effect of lowering
the acrylamide level appears to derive from the divalent or trivalent cation,
rather than from the
anion that is paired with it. Limitations to the cation/anion pair, other than
valence, are related to
their acceptability in foods, such as safety, solubility, and their effect on
taste, odor, appearance, and
texture. For example, the cation's effectiveness can be directly related to
its solubility. Highly
soluble salts, such as those salts comprising acetate or chloride anions, are
most preferred additives.
Less soluble salts, such as those salts comprising carbonate or hydroxide
anions can be made more
soluble by addition of phosphoric or citric acids or by disrupting the
cellular structure of the starch
based food. Suggested cations include calcium, magnesium, aluminum, iron,
copper, and zinc.
Suitable salts of these cations include calcium chloride, calcium citrate,
calcium lactate, calcium
malate, calcium gluconate, calcium phosphate, calcium acetate, calcium sodium
EDTA, calcium
glycerophosphate, calcium hydroxide, calcium lactobionate, calcium oxide,
calcium propionate,
calcium carbonate, calcium stearoyl lactate, magnesium chloride, magnesium
citrate, magnesium
lactate, magnesium malate, magnesium gluconate, magnesium phosphate, magnesium
hydroxide,
magnesium carbonate, magnesium sulfate, aluminum chloride hexahydrate,
aluminum chloride,
aluminum hydroxide, ammonium alum, potassium alum, sodium alum, aluminum
sulfate, ferric
chloride, ferrous gluconate, ferric ammonium citrate, ferric pyrophosphate,
ferrous fumarate, ferrous
lactate, ferrous sulfate, cupric chloride, cupric gluconate, cupric sulfate,
zinc gluconate, zinc oxide,
and zinc sulfate. The presently preferred embodiment of this invention uses
calcium chloride,
although it is believed that the requirements may be best met by a combination
of salts of one or
more of the appropriate cations. A number of the salts, such as calcium salts,
and in particular
calcium chloride, are relatively inexpensive and commonly used with certain
foods. Calcium
chloride can be used in combination with calcium citrate, thereby reducing the
collateral taste effects
of CaC12. Further, any number of calcium salts can be used in combination with
one or more
magnesium salts. One skilled in the art will understand that the specific
formulation of salts required
can be adjusted depending on the food product in question and the desired end-
product
characteristics.
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CA 02618225 2008-01-18
It should be understood that changes in the characteristics of the final
product, such as
changes in color, taste, and consistency can be adjusted by various means. For
example, color
characteristics in potato chips can be adjusted by controlling the amount of
sugars in the starting
product. Some flavor characteristics can be changed by the addition of various
flavoring agents to
the end product. The physical texture of the product can be adjusted by, for
example, the addition of
leavening agents or various emulsifiers.
XII. COMBINATIONS OF AGENTS IN MAKING DOUGH
In the above detailed embodiments of the invention, focus was on the reduction
of
acrylamide caused by a single agent, such as a divalent or trivalent cation or
one of several amino
acids, to lower the amount of acrylamide found in cooked snacks. Other
embodiments of the
invention involve the combination of various agents, such as combining calcium
chloride with other
agents to provide a significant reduction of acrylamide without greatly
altering the flavor of the
chips.
XIII. COMBINATIONS OF CALCIUM CHLORIDE, CITRIC ACID, PHOSPHORIC ACID
The inventors have found that calcium ions more effectively reduce acrylamide
content at
acidic pH. In the test shown below, the addition of calcium chloride in the
presence of an acid was
studied and compared to a sample with just the acid.
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Ingredient trol Phosphoric Phosphoric Citric Acid
Acid Acid & CaC12
& CaC12
Potato flakes/ 5490 5490 5490 5490
modified starch (g)
Sugar 360 360 360 360
Oil 90 90 90 90
Citric Acid 30
Phosphoric Acid 30 30
CaC12 30 30
Sodium bicarbonate 54
& monocalcium
phosphate
Emulsifier (g) 60 60 60 60
Total Dry Mix (g) 6000 6000 6000 6000
Water (ml) 3950 3950 3950 3950
Moisture % 2.16 2.34 2.07 1.60
Color L 67.69 71.39 72.70 73.27
A 5.13 3.24 1.62 0.95
B 26.51 26.91 26.05 26.24
Acrylamide (ppb) 1191 322 84 83
TABLE 15: Effect of Combining CaCl2 with Phosphoric Acid or Citric Acid on
Acrylamide
As seen in Table 15 above, the addition of phosphoric acid alone reduced the
acrylamide
formation by 73% while the addition of CaC12 and an acid dropped the
acrylamide level by 93%.
Figure 5 shows these results in graphical form. In this drawing, the
acrylamide level 502 of the
control is quite high (1191), but drops significantly when phosphoric acid
alone is added and even
lower when calcium chloride and an acid are added. At the same time, the
moisture levels 504 of the
various chips stayed in the same range, although it was somewhat lower in the
chips with added
agents. Thus, it has been demonstrated that calcium chloride and an acid can
effectively reduce
acrylamide.
Further tests were performed using calcium chloride and phosphoric acid as
additives to a
potato dough. Three different levels of calcium chloride were used,
corresponding to 0%, 0.45% and
0.90% by weight of the potato flakes. These were combined with three different
levels of phosphoric
acid, corresponding to 0%, 0.05%, or 0.1 % of the flakes. Additionally, three
levels of reducing sugar
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CA 02618225 2008-01-18
in the flakes were tested, corresponding to 0.2%, 1.07%, and 2.07%, although
not all combinations
of these levels are represented. Each test was mixed into dough, shaped, and
cooked to form potato
chips. The oil fry temperature, fry time, and sheet thickness were maintained
constant at 350F, 16
seconds, and 0.64 mm respectively. For clarity, the results are presented in
three separate tables
(16A,16B, and 16C) with each table showing the results for one of the levels
of sugar in the potato
flakes. Additionally, the tests are arranged so that the controls, with no
calcium chloride or
phosphoric acid, are on the left-hand side. Within the table, each level of
calcium chloride (CC) is
grouped together, with variations in the phosphoric acid (PA) following.
Cell Cntrl No 4.CC ICC TCC
CC No TPA IPA
IPA PA
(16) (5) (7) (4) (8)
CaC12 % --- --- 0.45 0.45 0.90
Phosphoric --- 0.05 --- 0.10 0.05
Acid%
Moisture 2.36 2.36 2.30 2.30 2.42
Oil 22.83 21.77 23.60 22.20 23.75
Color L 69.42 74.39 75.00 75.07 74.39
A 2.69 0.10 -0.02 -0.13 0.10
B 28.00 27.99 27.80 27.64 27.99
FA-crylamde 171 131 41 46 40
TABLE 16A: CaC12/Phosphoric Acid Effect on Acrylamide Level - 0.2% Reducing
Sugars
In the lowest level of reducing sugars in this test, we can see that the
levels of acrylamide
produced are normally in the lower range, as would be expected. At this level
of sugars, calcium
chloride alone dropped the level of acrylamide to less than 1/4 of the
control, with little additional
benefit gained by the addition of phosphoric acid. In the mid-range of
reducing sugars, shown in the
following table, the combination of calcium chloride reduces the level of
acrylamide from 367 ppb
in the control to 69 ppb in cell 12. Although some of this reduction may be
attributed to the slightly
higher moisture content of cell 12 (2.77 vs. 2.66 for the control), further
support is shown by the
significant reduction in acrylamide even when the levels of calcium chloride
and phosphoric acid are
halved. This is shown in cell 6, which has a significant reduction in
acrylamide and moisture content
lower than the control.
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CA 02618225 2008-01-18
Cell Cntrl No CC J,CC J,CC J,CC J,CC TCC TCC
TPA IPA IPA IPA J,PA 0 PA TPA
(15) (3) (2a) (2b) (6) (13) (9) (12)
CaC12 --- --- 0.45 0.45 0.45 0.45 0.90 0.90
Phosphoric Acid% --- 0.10 0.05 0.05 0.05 0.05 --- 0.10
Moisture 2.66 2.59 3.16 2.74 2.61 2.56 2.81 2.77
Oil 23.72 24.24 25.24 22.58 23.48 25.12 23.99 24.71
Color L 69.45 67.69 72.23 70.44 70.58 72.06 72.64 73.59
A 2.73 4.63 0.54 2.32 2.59 2.03 0.84 0.47
B 28.00 28.54 26.51 27.55 27.79 27.64 27.05 26.82
Acrylamide 367 451 96 170 192 207 39 69
TABLE 16B: CaC12/Phosphoric Acid Effect on Acrylamide Level - 1.07% Reducing
Sugars
Cell No CC J,CC ICC ICC J,CC TCC
IPA No PA No PA No PA TPA IPA
(11) (la) (lb) (lc) (10) (14)
CaC12 % --- 0.45 0.45 0.45 0.45 0.90
Phosphoric Acid% 0.05 --- --- --- 0.10 0.05
Moisture 2.47 2.68 2.60 3.19 2.80 3.18
Oil 24.70 25.07 24.48 22.81 24.19 23.25
Color L 61.84 62.32 63.86 69.42 69.11 72.61
A 8.10 5.18 6.70 3.00 3.78 1.28
B 28.32 26.27 28.00 27.66 27.70 26.78
Acrylamide 667 431 360 112 150 51
TABLE 16C: CaC12/Phosphoric Acid Effect on Acrylamide Level - 2.07% Reducing
Sugars
As can be seen from these three tables, the levels of calcium chloride and
phosphoric acid
necessary to reduce the level of acrylamide increases as the level of reducing
sugars increases, as
would be expected. Figure 6 shows a graph corresponding to the three tables
above, with the bars
602 showing acrylamide level and the points 604 demonstrating moisture level.
The results are again
grouped by the level of reducing sugar available from the potato; within each
group there is a general
movement downward as first one and then several acrylamide-reducing agents are
used to lower the
acrylamide level.
Several days later, another test with the same protocol as for the three
tables above was
conducted using only the potato flakes with 1.07% reducing sugars with the
same three levels of
calcium chloride and with four levels of phosphoric acid (0, 0.025%,0.05%, and
0.10%). The results
are shown below in Table 17. Figure 7 graphically shows the results for the
table, with acrylamide
levels expressed as bars 702 and calibrated to the markings on the left-hand
side while percentage
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CA 02618225 2008-01-18
moisture is expressed as points 704 and calibrated to the markings on the
right-hand side of the
drawing. As the amount of calcium chloride increases, e.g. moving from left to
right across the
whole table, the acrylamide decreases. Likewise, for each level of calcium
chloride, e.g. moving left
to right within one level of calcium chloride, the level of acrylamide also
generally decreases.
Cell Cntrl No CC No CC ICC I.CC TCC TCC TCC
IPA TPA IPA IPA JJPA IPA TPA
(1) (4) (7) (3) (6) (8) (2) 5)
CaC12 --- --- --- 0.45 0.45 0.90 0.90 0.90
Phosphoric --- 0.050 0.100 0.050 0.050 0.025 0.050 0.100
Acid%
Moisture 2.68 2.52 2.38 2.29 2.55 2.45 2.78 2.61
Oil 23.74 22.57 22.13 24.33 23.84 22.54 24.11 22.73
Color L 65.97 64.67 64.55 65.18 66.82 68.36 70.23 68.75
A 4.75 5.23 5.53 5.06 4.09 3.17 2.19 2.92
B 27.70 27.83 27.94 27.79 27.64 27.17 26.28 27.06
Acrylamide 454 435 344 188 77 233 80 66
TABLE 17: CaC12 / Phosphoric Acid Effect on Acrylamide Level - 1.07% Reducing
Sugars
XIV. CALCIUM CHLORIDE/CITRIC ACID WITH CYSTEINE
In some of the previous tests on corn chips performed by the inventors, the
amount of
calcium chloride and phosphoric acid necessary to bring the level of
acrylamide to a desired level
produced objectionable flavors. The following test was designed to reveal if
the addition to the
potato dough of cysteine - which has been shown to lower the levels of
acrylamide in the chips -
would allow the levels of calcium chloride and acid to be lowered to
acceptable taste levels while
keeping the level of acrylamide low. In this test, the three agents were added
to the masa (dough) at a
ratio of (i.) 0.106% Ca/C12, 0.084% citric acid, and 0.005% L. cysteine in a
first experiment; (ii)
0.106% Ca/C12 and 0.084% citric acid, but no cysteine in a second experiment,
and 0.053% Ca/C12,
0.042% citric acid with 0.005% L. cysteine as a third experiment. Each
experiment was duplicated
and run again, with both results shown below. The masa is about 50% moisture,
so the
concentrations would approximately double if one translates these ratios to
solids only. Additionally,
in each test, part of the run was flavored with a nacho cheese seasoning at
about 10% of the base
chip weight. Results of this test are shown in Table 18 below. In this table,
for each category of chip,
e.g., plain chip, control, the results of the first-run experiment are given
in acrylamide #1; the results
of the second experiment are given as acrylamide #2, and the average of the
two given as acrylamide
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CA 02618225 2008-01-18
average. Only one moisture level was taken, in the first experiment; that
value is shown.
Cell Plain chip Nacho chip
Cntrl TCC T'CC J,CC Cntrl TCC TCC J,CC
TCitric TCitric 1Citric TCitric TCitric JCitric
0 Cys Cys Cys 0 Cys Cys Cys
CaC12 (%) 0.106 0.106 0.053 0.106 0.106 0.053
Citric acid (%) 0.084 0.084 0.042 0.084 0.084 0.042
Cysteine (%) 0.005 0.005 0.005 0.005
Acrylamide #1 ppb 163 154 70 171 90 55 62 77
Acrylamide #2 ppb 102 113 74 103 71 53 50 76
Acrylamide average 132.5 133.5 72 137 80.5 54 56 76.5
ppb
Moisture % 1.07 0.91 1.07 0.95 1.26 1.49 1.23 1.25
TABLE 18: Effect of Cysteine with CaC12 / Citric Acid on Acrylamide Level in
Corn Chips
When combined with 0.106% CaC12 and 0.084% citric acid, the addition of
cysteine cut the
production of acrylamide approximately in half. In the chips flavored with
nacho flavoring, the
calcium chloride and citric acid alone reduced the production of acrylamide
from 80.5 to 54 ppb,
although in this set of tests, the addition of cysteine did not appear to
provide a further reduction of
acrylamide.
Figure 8 graphically presents the same data as the table above. For each type
of chip on
which the experiment was run (e.g., plain chip, control), two bars 802 show
the acrylamide results.
Acrylamide results 802a from the first experiment are shown on the left for
each type chip, with the
acrylamide results 802b from the second experiment shown on the right. Both
acrylamide results are
calibrated to the markings on the left of the graph. The single moisture level
is shown as a point 804
overlying the acrylamide graph and is calibrated to the markings on the right
of the graph.
After the above test was completed, fabricated potato chips were similarly
tested, using
potato flakes containing two different levels of reducing sugars. To translate
the concentrations used
in the corn chip test to fabricated potato chips, the sum of the potato
flakes, potato starch, emulsifiers
and added sugar were considered as the solids. The amounts of CaC12, citric
acid, and cysteine were
adjusted to yield the same concentration as in the corn chips on a solids
basis. In this test, however,
when higher levels of calcium chloride and citric acid were used, a higher
level of cysteine was also
used. Additionally, a comparison was made in the lower reducing sugar portion
of the test, to the use
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CA 02618225 2008-01-18
of calcium chloride in combination with phosphoric acid, with and without
cysteine. The results are
shown in Table 19.
We can see from these that in potato flakes with 1.25% of reducing sugars, the
combination
of calcium chloride, citric acid, and cysteine at the first level above
reduced the formation of
acrylamide from 1290 ppb to 594 ppb, less than half of the control figure.
Using the higher levels of
the combination of agents reduced the formation of acrylamide to 306 ppb, less
than half of the
control amount.
Using the same potato flakes, phosphoric acid and calcium chloride alone
reduced the
formation of acrylamide from the same 1290 to 366 ppb, while a small amount of
cysteine added
with the phosphoric acid and calcium chloride reduced the acrylamide still
further, to 188 ppb.
Finally, in the potato flakes having 2% reducing sugars, the addition of
calcium chloride,
citric acid, and cysteine reduced the formation of acrylamide from 1420 to 665
ppb, less than half.
Cell Medium reducing sugars High reducing sugars (2%)
(1.25%)
Cntrl J,CC TCC CC CC Cntrl ,ACC
ICitric TCitric PhosA PhosA ,Citric
,Cyst TCyst OCyst Cyst ,Cyst
(1B) (2) (3) (4) (4A) (6) (7)
Calcium chloride 10.2 20.4 36 36 10.2
Citric acid 8 16 8
Phosphoric acid 4 4
Cysteine 0.48 0.96 0.48 0.48
Acrylamide ppb 1290 594 306 366 188 1420 665
Moisture % 1.82 2.06 2.12 2.06 2.33 2.28 2.23
Color L 56.84 65.47 69.29 66.88 73.09 61.06 63.50
A 10.20 6.42 4.07 4.42 1.55 9.03 7.93
B 27.53 28.40 28.17 28.10 27.07 28.07 28.00
TABLE 19: Effect of Cysteine with CaCl2 / Acid on Acrylamide Level in Potato
Chips
Figure 9 demonstrates graphically the results of this experiment. Results are
shown grouped
first by the level of reducing sugars, then by the amount of acrylamide-
reducing agents added. As in
the previous graphs, bars 902 representing the level of acrylamide are
calibrated according to the
markings on the left-hand side of the graph, while the points 904 representing
the moisture level are
calibrated according to the markings to the right-hand side of the graph.
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CA 02618225 2008-01-18
The above experiments have shown that the acrylamide-reducing agents do not
have to be
used separately, but can be combined to provide added benefit. This added
benefit can be used to
achieve increasingly lower levels of acrylamide in foods or to achieve a low
level of acrylamide
without producing significant changes to the taste of texture of those foods.
Although the specific
embodiments shown have disclosed calcium chloride combined with citric acid or
phosphoric acid
and these with cysteine, one of ordinary skill in the art would realize that
the combinations could use
other calcium salts, the salts of other divalent or trivalent cations, other
food-grade acids, and any of
the other amino acids that have been shown to lower acrylamide in a finished
food product.
Additionally, although this has been demonstrated in potato chips and corn
chips, one of ordinary
skill in the art would understand that the same use of combinations of agents
can be used in other
fabricated food products that are subject to the formation of acrylamide, such
as cookies, crackers,
etc.
XV. AGENTS TO REDUCE ACRYLAMIDE ADDED IN THE MANUFACTURE OF POTATO FLAKES
The addition of calcium chloride and an acid has been shown to lower
acrylamide in fried
and baked snack foods formulated with potato flakes. It is believed that the
presence of an acid
achieves its effect by lowering the pH. It is not known whether the calcium
chloride interferes with
the loss of the carboxyl group or the subsequent loss of the amine group from
free asparagine to
form acrylamide. The loss of the amine group appears to require high
temperature, which generally
occurs toward the end of the snack dehydration. The loss of the carboxyl group
is believed to occur
at lower temperatures in the presence of water.
Potato flakes can be made either with a series of water and steam cooks
(conventional) or
with a steam cook only (which leaches less from the exposed surfaces of the
potato). The cooked
potatoes are then mashed and drum dried. Analysis of flakes has revealed very
low acrylamide levels
in flakes (less than 100 ppb), although the products made from these flakes
can attain much higher
levels of acrylamide.
It was theorized that if either lowering dough pH with acid or adding calcium
chloride to the
dough interferes with the loss of the carboxylic group, then introducing these
additives during the
flake production process might either (a) reduce the carboxyl loss thus
reducing the rate of amine
loss during the snack food dehydration or (b) whatever the mechanism, insure
that the intervention
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CA 02618225 2008-01-18
additive is well distributed in the dough that is dehydrated into the snack
food. The former, if it
happens, would be a likely bigger effect on acrylamide than the latter.
Another possible additive to reduce the formation of acrylamide in fabricated
food products
is asparaginase. Asparaginase is known to decompose asparagine to aspartic
acid and ammonia. The
process of making flakes by cooking and mashing potatoes (a food ingredient)
breaks down the cell
walls and provides an opportunity for asparaginase to work. In a preferred
embodiment, the
asparaginase is added to the food ingredient in a pure form as food grade
asparaginase either as a
powder or in an aqueous solution. Asparaginase can be combined with other
acrylamide-reducing
agents discussed herein, such as amino acids and di- and trivalent cations.
The inventors designed the following sets of experiments to study the
effectiveness of
various agents added during the production of the potato flakes in reducing
the level of acrylamide in
products made with the potato flakes.
XVI. CALCIUM CHLORIDE AND PHOSPHORIC ACID USED IN MAKING POTATO FLAKES
This series of tests were designed to evaluate the reduction in the level of
acrylamide when
CaC12 and/or phosphoric acid are added during the production of the potato
flakes. The tests also
address whether these additives had the same effect as when they are added at
the later stage of
making the dough.
For this test, the potatoes comprised 20% solids and 1% reducing sugar. The
potatoes were
cooked for 16 minutes and mashed with added ingredients. All batches received
13.7 gm of an
emulsifier and 0.4 gm of citric acid. Four of the six batches had phosphoric
acid added at one of two
levels (0.2% and 0.4% of potato solids) and three of the four batches received
CaC12 at one of two
levels (0.45% and 0.90% of the weight of potato solids). After the potatoes
were dried and ground
into flakes of a given size, various measurements were performed and each
batch was made into
dough. The dough used 4629 gm of potato flakes and potato starch, 56 gm of
emulsifier, 162 ml of
liquid sucrose and 2300 ml of water. Additionally, of the two batches that did
not receive phosphoric
acid or CaC12 during flake production, both batches received these additives
at the given levels as the
dough was made. The dough was rolled to a thickness of 0.64 mm, cut into
pieces, and fried at 350 F
for 20 seconds. Table 20 below shows the results of the tests for these
various batches.
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CA 02618225 2010-04-07
Batch 0 Ca 4Ca ICa 'RCa 1'Ca 'R Ca
4phos lphos 4phos lphos J-phos tphos
(C) in (B) in (F) in (A) in (D) in (E) in
flakes flakes dough flakes dough flakes
Added to flakes
Wt. (gm) Calcium Chloride 0 24.7 0 49.4 0 49.4
Wt. (gm) Phosphoric Acid 11.0 11.0 0 11.0 0 21.9
Dried Flake Tests
Moisture (%) 6.3 6.5 4.5 6.8 6.2 7.7
Water Absorption Index (WAI) 8.2 8.3 9.2 8.2 8.1 8.1
(%)
On 20 mesh 1.5 1.8 2.0 1.0 1.7 1.6
On 40 mesh 26.6 30.9 32.3 27.2 28.3 24.4
On 60 mesh 35.3 37.1 36.1 38.4 37.5 35.3
On 80 mesh 14.6 13.2 12.0 14.5 14.4 16.0
On 100 mesh 5.7 4.8 4.5 5.4 5.4 6.5
On 200 mesh 11.5 8.8 8.6 10.1 9.3 12.1
Through 200 mesh 4.7 3.3 4.5 3.4 3.3 4.0
Added to dough
Calcium Chloride dihydrate 0 0 23.7 0 47.4 0
Phosphoric Acid 0 0 14.4 0 7.9 0
Test Results on Chips
Moisture 1.87 2.04 2.04 2.07 1.97 2.05
Oil 23.53 23.82 25.12 23.76 24.44 24.98
Color - L 54.63 62.58 67.28 66.89 69.48 66.87
Color - A 13.63 9.23 6.99 6.27 5.61 7.21
Color - B 27.32 28.59 29.54 28.85 29.26 29.37
Acrylamide 1286 344 252 129 191 141
TABLE 20: Effect of CaC12 / Phosphoric Acid added to Flakes or Dough on
Acrylamide Level
As seen in the results above and in the accompanying graph of Figure 10, the
acrylamide
level represented by numerals 1002 was the highest in Test C when only
phosphoric acid was added
to the flake preparation and was the lowest when calcium chloride and
phosphoric acid were used in
combination. Numerals 1004 represent the moisture content of the fried chip.
XVIL ASPARAGINASE USED IN MAKING POTATO FLAKES
Asparaginase is an enzyme that decomposes asparagine to aspartic acid and
ammonia. Since
aspartic acid does not form acrylamide, the inventors reasoned that
asparaginase treatment should
reduce acrylamide formation when the potato flakes are heated.
The following test was performed. Two grams of standard potato flakes was
mixed with 35
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CA 02618225 2008-01-18
ml of water in a metal drying pan. The pan was covered and heated at 100 C for
60 minutes. After
cooling, 250 units of asparaginase in 5 ml water were added, an amount of
asparaginase that is
significantly more than the calculated amount necessary. Enzymes are sold in
units of activity. One
unit of activity is defined as follows: One unit will liberate 1.0 mole of
ammonia from L-
asparagine per minute at pH 8.6 at 37 C. For control, potato flakes and 5 ml
of water without
enzyme was mixed. The potato flakes with asparaginase were held at room
temperature for 1 hour.
After enzyme treatment, the potato flake slurry was dried at 60 C overnight.
The pans with dried
potato flakes were covered and heated at 120 C for 40 minutes. Acrylamide was
measured by gas
chromatograph, mass spectrometry of brominated derivative. The control flakes
contained 11,036
ppb of acrylamide, while the asparaginase-treated flakes contained 117 ppb of
acrylamide, a
reduction of more than 98%.
Following this first test, investigation was made into whether or not it was
necessary to cook
the potato flakes and water prior to adding asparaginase for the enzyme to be
effective. To test this,
the following experiment was performed:
Potato flakes were pretreated in one of four ways. In each of the four groups,
2 grams of
potato flakes were mixed with 35 milliliters of water. In the control pre-
treatment group (a), the
potato flakes and water were mixed to form a paste. In group (b), the potato
flakes were
homogenized with 25 ml of water in a Bio Homogenizer M 133/1281-0 at high
speed and mixed
with an additional 10 ml of deionized water. In group (c), the potato flakes
and water were mixed,
covered, and heated at 60 C for 60 minutes. In group (d), the potato flakes
and water were mixed,
covered, and heated at 100 C for 60 minutes. For each pre-treatment group (a),
(b), (c), and (d), the
flakes were divided, with half of the pre-treatment group being treated with
asparaginase while the
other half served as controls, with no added asparaginase.
A solution of asparaginase was prepared by dissolving 1000 units in 40
milliliters of
deionized water. The asparaginase was from Erwinia chrysanthemi, Sigma A-2925
EC 3.5.1.1. Five
milliliters of asparaginase solution (5ml) was added to each of the test
potato flake slurries (a), (b),
(c), and (d). Five milliliters of deioninzed water was added to the control
potato flake slurry (a). All
slurries were left at room temperature for one hour, with all tests being
performed in duplicate. The
uncovered pans containing the potato flake slurries were left overnight to dry
at 60 C. After covering
the pans, the potato flakes were heated at 120 C for 40 minutes. Acrylamide
was measured by gas
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CA 02618225 2008-01-18
chromatography, mass spectroscopy of brominated derivative.
As shown in Table 21 below, asparaginase treatment reduced acrylamide
formation by more
than 98% for all pretreatments. Neither homogenizing nor heating the potato
flakes before adding the
enzyme increased the effectiveness of asparaginase. In potato flakes,
asparagine is accessible to
asparaginase without treatments to further damage cell structure. Notably, the
amount of
asparaginase used to treat the potato flakes was in large excess. If potato
flakes contain 1%
asparagine, adding 125 units of asparaginase to 2 grams of potato flakes for 1
hour is approximately
a 50-fold excess of enzyme.
Acrylamide ppb Acrylamide as % of Control
Control - No Test - Asparaginase
Pre-treatment Asparaginase
(a) No pre-treatment 12512 107 0.9
(b) Homogenizing 12216 126 1.0
(c) Heated at 60 C 12879 105 0.8
(d) Heated at 100 C 12696 166 1.3
TABLE 21: Effect of Pretreatments of Potato Flakes on Effectiveness of
Asparagine
Another set of tests was designed to evaluate whether the addition of
asparaginase during the
production of potato flakes provides a reduction of acrylamide in the cooked
product made from the
flakes and whether buffering the mashed potatoes used to make the flakes to a
preferred pH for
enzyme activity (e.g., pH = 8.6) increases the effectiveness of the
asparaginase. The buffering was
done with a solution of sodium hydroxide, made with four grams of sodium
hydroxide added to one
liter of water to form a tenth molar solution.
Two batches of potato flakes were made as controls, one buffered and one un-
buffered.
Asparaginase was added to two additional batches of potato flakes; again one
was buffered while the
other was not. The asparaginase was obtained from Sigma Chemical and was mixed
with water in a
ratio of 8 to 1 water to enzyme. For the two batches in which asparaginase was
added, the mash was
held for 40 minutes after adding the enzyme, in a covered container to
minimize dehydration and
held at approximately 36 C. The mash was then processed on a drum dryer to
produce the flakes.
The potato flakes were used to make potato dough according to the previously
shown protocols, with
the results shown in Table 22 below.
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CA 02618225 2008-01-18
Measurement Unbuffered Unbuffered Buffered Buffered
Control Asparaginase Control Asparaginase
Moisture 1.56 1.53 1.68 1.61
Oil 22.74 23.12 21.77 21.13
Color - L 61.24 60.70 57.24 57.35
Color - A 6.57 9.30 5.04 7.52
Color - B 28.95 28.29 27.12 27.41
Acrylamide ppb 768 54 1199 111
TABLE 22: Effect of Asparaginase and Buffering on Acrylamide Level in Potato
Chips
As shown in Table 22, the addition of asparaginase without a buffer reduced
the production
of acrylamide in the finished chips from 768 to 54 ppb, a reduction of 93%.
The use of a buffer did
not appear to have the desired effect on the formation of acrylamide; rather
the use of the buffered
solution allowed a greater amount of acrylamide to form in both the control
and the asparaginase
experiments. Still, the asparaginase reduced the level of acrylamide from 1199
to 111, a reduction of
91 %. Figure 11 shows the results from Table 22 in a graphical manner. As in
the previous drawings,
bars 1102 represent the level of acrylamide for each experiment, calibrated
according to the
markings on the left-hand side of the graph, while points 1104 represent the
moisture level in the
chips a, calibrated according to the markings on the right-hand side of the
graph.
Tests were also run on the samples to check for free asparagine to determine
if the enzyme
was active. The results are shown below in Table 23.
Control Asparaginase Control Asparaginase
Unbuffered Unbuffered Buffered Buffered
Free Asparagine 1.71 0.061 2.55 0.027
Fructose <0.01 <0.01 <0.01 <0.01
Glucose <0.02 <0.02 <0.02 <0.02
Sucrose 0.798 0.828 0.720 0.322
TABLE 23: Test for Free Asparagine in Enzyme Treated Flakes
In the unbuffered group, the addition of asparaginase reduced the free
asparagine from
1.71 to 0.061, a reduction of 96.5%. In the buffered group, the addition of
asparaginase reduced
the free asparagine from 2.55 to 0.027, a reduction of 98.9%.
Finally, sample flakes from each group were evaluated in a model system. In
this model
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CA 02618225 2008-01-18
system, a small amount of flakes from each sample was mixed with water to form
an approximate
50% solution of flakes to water. This solution was heated in a test tube for
40 minutes at 120 C. The
sample was then analyzed for acrylamide formation, with the results shown in
Table 24. Duplicate
results for each category are shown side by side. In the model system, the
addition of asparaginase to
the unbuffered flakes reduced the acrylamide from an average of 993.5 ppb to
83 ppb, a reduction of
91.7%. The addition of asparaginase to the buffered flakes reduced the
acrylamide from an average
of 889.5 ppb to an average of 64.5, a reduction of 92.7%.
Control Asparaginase Control Asparaginase
Unbuffered Unbuffered Buffered Buffered
Ac lamide p b 1019 968 84 82 960 819 70 59
TABLE 24: Model System Effect of Asparaginase on Acrylamide
XVIII. ROSEMARY EXTRACT ADDED TO FRYING OIL
In a separate test, the effect of adding rosemary extract to the frying oil
for fabricated potato
chips was examined. In this test, identically fabricated potato chips were
fried either in oil that had
no additives (controls) or in oil that had rosemary extract added at one of
four levels: 500, 750,
1,000, or 1,500 parts per million. Table 25 below gives the results of this
test.
Level of Rosemary p pm 0 0 500 750 1,000 1,500
Moisture % 2.58 2.64 2.6
Acrylamide p b 1210 1057 840 775 1211 1608
TABLE 25: Effect of Rosemary on Acrylamide
The average acrylamide level in the control chips was 1133.5 ppb. Adding 500
parts per
million of rosemary to the frying oil reduced the acrylamide to 840, a
reduction of 26%, while
increasing the rosemary to 750 parts per million reduced the formation of
acrylamide further, to 775,
a reduction of 31.6%. However, increasing the rosemary to 1000 parts per
million had no effect and
increasing rosemary to 1500 parts per million caused the formation of
acrylamide to increase to 1608
parts per billion, an increase of 41.9%.
Figure 12 demonstrates the results of the rosemary experiment graphically. As
in the
previous examples, the bars 1202 demonstrate the level of acrylamide and are
calibrated to the
divisions on the left-hand side of the graph, while the points 1204
demonstrate the amount of
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moisture in the chips and are calibrated to the divisions on the right-hand
side of the graph.
The disclosed test results have added to the knowledge of acrylamide-reducing
agents that
can be used in thermally processed, fabricated foods. Divalent and trivalent
cations, the enzyme
asparaginase, and amino acids have been shown to be effective in reducing the
incidence of
acrylamide in thermally processed, fabricated foods. These agents can be used
individually, but can
also be used in combination with each other or with acids that increase their
effectiveness. The
combination of agents can be utilized to further drive down the incidence of
acrylamide in thermally
processed foods from that attainable by single agents or the combinations can
be utilized to attain a
low level of acrylamide without undue alterations in the taste and texture of
the food product.
Asparaginase has been tested as an effective acrylamide-reducing agent in
fabricated foods. It has
also been shown that these agents can be effective not only when added to the
dough for the
fabricated food, but the agents can also be added to intermediate products,
such as dried potato
flakes or other dried potato products, during their manufacture. The benefit
from agents added to
intermediate products can be as effective as those added to the dough.
XIX. EFFECT OF ACRYLAMIDE-REDUCING AGENT HAVING A FREE THIOL ON ACRYLAMIDE
FORMATION
Another embodiment of the invention involves reducing the production of
acrylamide by the
addition of a reducing agent with a free thiol compound to a snack food dough
prior to cooking or
thermal processing. As used herein, a free thiol compound is an acrylamide
reducing agent having a
free thiol. As previously discussed, it is believed that the free thiol of
cysteine can react with the
double carbon bond of acrylamide and act as an inhibitor of the Maillard
reaction.
A test was conducted to confirm the free thiol is likely responsible for the
acrylamide
reduction. Five free thiol compounds were prepared in equimolar basis, each
compound having a
concentration 6.48 mmoles per liter in a 0.5 molar sodium phosphate buffer
having pH of 7.0 with
0.4% asparagine (30.3 millimolar) and 0.8% glucose (44.4 millimolars). A
control sample having no
free thiol compounds was also prepared. The six solutions were each heated at
120 C for 40 minutes.
The solutions were then measured for acrylamide concentrations. The results
are shown in Table 26
below:
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Compound Acrylamide (p b) As % of Control
Control (No Free Thiol) 4146 100
Cysteine ("L-Cysteine") 1128 27
N-Acetyl-L-Cysteine 1231 30
N-Acetyl-cysteamine 1204 29
Glutathione Reduced 1153 28
Di-thiothreitol 1462 35
TABLE 26: Effect of Free Thiol Compounds on Acrylamide Reduction Through
Decomposition
The above experiment confirms that it is the free thiol group that reduces
acrylamide. The
free amino group of cysteine does not contribute to reducing acrylamide
because N-acetyl-L-
cysteine having a blocked amino group is about as effective as cysteine. The
carboxyl group of
cysteine does not contribute to reducing acrylamide because N-acetyl-
cysteamine, which has no
carboxyl group is about as effective as cysteine at reducing acrylamide.
Gluthathione, a tripeptide
with cysteine in the middle position, was equivalent to cysteine. Although
dithiothreitol has two
thiol groups, acrylamide with dithiothreitol was similar to the compounds with
one thiol group. The
two thiol groups in dithiothreitol may react to from disulfides so
dithiothreitol was less effective on
an equal molar basis than the other thiol containing compounds.
Experimentation, as exemplified by Table 26 above, has shown that acrylamide
reduction is
roughly proportional to the concentration of added free thiols, such as
cysteine. However, collateral
effects on the characteristics, such as color, taste, and texture of the final
product from the addition
of a free thiol compound as cysteine must be considered. High levels of
cysteine, for example, can
impart undesirable off-flavors in the final product. Hence, additives that can
increase or magnify the
effectiveness of a free thiol compound, such as cysteine, are desirable
because such additives can
permit the same level of acrylamide reduction with a lesser concentration of a
thiol compound. It
has been discovered that when a reducing agent is added to a free thiol
compound such as cysteine,
acrylamide reduction is enhanced. Reducing agents are known in oxidation-
reduction chemistry to
be compounds that are electron donors and oxidizing agents are known to be
electron acceptors.
XX. EFFECT OF CYSTEINE + REDUCING AGENT ON ACRYLAMIDE DECOMPOSITION
Simple model systems can be used to test the magnified effectiveness of free
thiol
compounds with the addition of a reducing agent. A control sample solution
comprising a free thiol
(1.114 millimolar of cysteine) and acrylamide (0.0352 millimolar) was prepared
in a 0.5 molar
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sodium phosphate buffer having a pH of 7Ø The solution was heated at 120 C
for 40 minutes. The
recovery of the added acrylamide was 21 %. Hence, the amount of acrylamide
reduction for the
control sample with no reducing agent was 79%. Even though the molar ratio of
cysteine to
acrylamide was more than 30, not all of the acrylamide reacted with cysteine.
A test was then run with free thiol compounds and a reducing agent. A solution
comprising
135 ppm of a free thiol compound (1.114 millimolar of cysteine), 2500 ppb
acrylamide (0.0352
millimolars), and about 305 ppm reducing agent (1.35 millimolar of stannuous
chloride dihydrate)
was prepared in a 0.5 molar sodium phosphate buffer having a pH of 7Ø After
heating at 120 C for
40 minutes, the recovery of added acrylamide was measured to be less than 4%.
Hence, the amount
of acrylamide reduction with the sample containing a reducing agent was over
96%, an additional
17% over the free thiol alone, or control sample.
XXI. EFFECT OF CYSTEINE + OXIDIZING AGENT ON ACRYLAMIDE DECOMPOSITION
A test was then run with the addition of an oxidizing agent instead of a
reducing agent. A
solution of 135 ppm of a free thiol (1.114 millimolar of cysteine), 2500 ppb
of acrylamide (0.0352
millimolars), and a 235 ppm of an oxidizing agent (1.35 millimolars of
dehydroascorbic acid) was
prepared in a 0.5 molar solution of sodium phosphate buffer having a pH of
7Ø After heating at
120 C for 40 minutes, the recovery of added acrylamide was measured to be
about 27%. Hence, the
amount of acrylamide reduction with the sample containing the oxidizing agent
was about 73%,
which is less then the reduction achieved by the cysteine control sample.
Thus, acrylamide
decomposition worsened with the addition of the oxidizing agent.
Further tests were conducted with other oxidizing and reducing agents with an
acrylamide
solution having about 2500 ng/ml, or 2500 ppb of acrylamide. The results are
provided in Table 27
below.
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Compound Concentration Concentration Recovery of % Recovery of
(u ml) millimolar Ac lamide ng/ml Acrylamide
Control Sample (Free Thiol Only)
Cysteine 135 1.114 534 21 %
Reducing Agent + 135 ppm of Cysteine
Ascorbic Acid 11.4 9%
(Vitamin C)
Stannous 304.6 1.350 68 3%
chloride
dihydrate
Sodium sulfite 170.2 1.350 69 3%
Sodium meta- 256.6 1.350 24 1%
bisulfite
Oxidizing Agent + 135 p pm of Cysteine
Dehydroascorbic 235 1.350 673 27%
acid
Gallic acid 253.9 1.350 1111 44%
monohydrate
Catechin hydrate 391.9 1.350 877 35%
Epicatechin 391.9 1.350 827 33%
Rutin hydrate 824.2 1.350 1306 52%
TABLE 27: Effect of Oxidizing and Reducing Agents With Cysteine on Acrylamide
Figure 13 graphically illustrates the theorized effect of the addition of an
oxidizing or
reducing agent to an acrylamide-reducing agent. Without being bound to theory,
it is believed that
the reducing agents 1304 increase or magnify the effectiveness of cysteine by
keeping cysteine in the
reduced, thiol 1306 form. As discussed above, it is believed that the free
thiol of cysteine reacts with
the double bond of acrylamide. An oxidizing agent 1302, such as
dehydroascorbic acid, likely
converts the cysteine thiol 1306 into an inactive cysteine disulfide (cystine)
1308. In one
embodiment of the invention, the reducing agent having a standard reduction
potential (E ) of
between about +0.2 and -2.0 volts is used.
XXII. ENHANCED EFFECT OF THIOL WITH A REDUCING AGENT WITH POTATO FLAKES
A test was performed to compare the reduction of acrylamide with a free thiol
with and
without a reducing agent in the presence of potato flakes. Six vials were
prepared having 3 grams of
potato flakes mixed with 3 mL of dionized water. Cysteine was added to the
vials at concentrations
(ug cysteine/g potato flake) of 800 ppm, 400 ppm, 200 ppm, and 100 ppm.
Casein, a potential free
thiol source, was added to a vial at the 1 % level. The six samples were each
heated at 120 C for 40
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minutes. The solutions were then measured for acrylamide concentrations. The
results are shown in
Table 28 below:
Sample Added Cysteine Acrylamide (ppb) Acrylamide as a % of
(ppm) Control
Control Potato Flakes 0 2695 100
Cysteine 800 2220 82
Cysteine 400 2179 81
Cysteine 200 2612 97
Cysteine 100 2832 105
Casein (1%) 2808 104
TABLE 28: Effect of Various Concentration Levels on Acrylamide Reduction
without a Reducing
Agent
The data again confirms that as the concentration of cysteine increases, the
acrylamide
reduction also increases. The above test also indicates that 1 % Casein
without a reducing agent does
not reduce acrylamide.
As shown in Table 27 above, sodium sulfite (reducing agent) increased the
effectiveness of
cysteine in decreasing added acrylamide an additional 18% over the free thiol,
or control sample. A
test was conducted to determine the effect of sodium sulfite on the
effectiveness of cysteine and
casein in decreasing acrylamide levels in potato flakes. Five vials were
prepared having 3 grams of
potato flakes mixed with 3 mL of dionized water. Cysteine was added to two
vials at a
concentration of 400 ppm (ug cysteine/g potato flake). Casein was added to a
vial at the 1% level.
Sodium sulfite was added at 483 ppm (ug sulfur dioxide per g of potato flake)
to the casein vial and
one of the cysteine vials. The samples were each heated at 120 C for 40
minutes. The solutions
were then measured for acrylamide concentrations. The results are shown in
Table 29 below:
Thiol Reducing Agent Acrylamide Acrylamide as %
(p b of Control
0 ppm Cysteine -- 3567 100
(Control)
400 ppm Cysteine -- 2500 70
-- 483 p pm sodium sulfite 3004 84
400 ppm cysteine 483 m sodium sulfite 2351 66
1 % Casein 483 p pm sodium sulfite 2632 74
TABLE 29: Effect of Various Concentration Levels on Acrylamide Reduction of
Potato Flakes
without a Reducing Agent
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Table 28 indicates that a 1% Casein addition failed to reduce acrylamide
levels in potato
flakes without a reducing agent. Table 29, however, reveals that the addition
of a reducing agent
(483 ppm sodium sulfite) results in an additional 10% acrylamide reduction
over the sodium sulfite
alone.
The thiol and reducing agent were less effective in reducing acrylamide levels
in the potato
flakes samples (Table 28 and 29) than in the non-potato flakes solutions.
There are several potential
reasons that explain this. For example, acrylamide was added in the non-potato
flake samples but
had to be formed in the potato flake samples. Thus, acrylamide formation was
probably more
important than decomposition. Further, conditions were not optimized for
potato flakes. The pH of
the potato flakes was not adjusted to pH 7, which would increase the
reactivity of cysteine with
acrylamide.
In one embodiment, the free thiol compound 1306 is selected from the group
consisting of
cysteine, N-acetyl-L-cysteine, N-acetyl-cysteamine, glutathione reduced,
dithiothreitol, casein, and
combinations thereof. In one embodiment, the reducing agent 1304 is selected
from the group
consisting of stannous chloride dihydrate, sodium sulfite, sodium meta-
bisulfite, ascorbic acid,
ascorbic acid derivatives, isoascorbic acid (erythorbic acid), salts of
ascorbic acid derivatives, iron,
zinc, ferrous ions, and combinations thereof.
One advantage of the present invention is that the same reduction of
acrylamide can be
achieved by using less free thiol when the free thiol compound is mixed with a
reducing agent.
Thus, undesirable off-flavors can be reduced or eliminated. The acrylamide
reduction can be
achieved by using free thiol compound and reducing agent in any dough-based
snack food. Another
benefit of the present invention is the inherent nutritional benefit
associated with some reducing
agents. Ascorbic acid, for example, is also commonly known as vitamin C.
XXIII. ADDITIONAL EXAMPLES OF ASPARAGINASE USE IN FABRICATED SNACKS
Applicants have previously discussed and disclosed examples of the use of the
enzyme
asparaginase with fabricated foods as an acrylamide reducing agent. The
following are additional
examples of such practice that illustrate the utility and flexibility of this
approach.
In a first example, corn is cooked to a moisture level of 45%. The corn is
milled with the
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addition of water and, except for the control samples, the enzyme
asparaginase, in order to bring the
water level to 50%. A masa was formed for each test run under the conditions
detailed in the
"Description" column below in Table 30. After the masa was prepared pursuant
to the conditions
listed in the "Description" column, samples were removed and allowed to set
for 3, 6, or 9 minutes
before being quenched with an alcohol solution. This alcohol solution
deactivates the asparaginase
enzyme, thus simulating a dwell-time for the enzyme in the masa after mixing.
The simulated dwell-
time for each test run is reflected in the "Set Time" column of Table 30.
After the quenching, each
sample is then tested for the level of asparagine, and the results of these
tests are also reflected in
Table 30. After the test runs were performed, the masa was formed into a chip,
the chip was fried to
a moisture level of 1.1 %, and the level of acrylamide found in each chip was
measured. The level of
acrylamide detected after frying to this moisture level was found to
correspond linearly to the
amount of asparagine measured after each test run as previously described.
Table 30 below provides
for the protocol for each test run and the results.
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Test Run Set Time (mins) Description Asparagine
n-Mole
1 Control 5.16
2 3 120 units asparaginase/kg of 3.65
masa added with water at pH
8.5 and ambient temperature.
3 6 120 units asparaginase/kg of 2.69
masa added with water at pH
8.5 and ambient temperature.
4 9 120 units asparaginase/kg of 1.31
masa added with water at pH
8.5 and ambient temperature.
3 120 units asparaginase/kg of 2.99
masa added with water at pH
8.5 and 60 F.
6 6 120 units asparaginase/kg of 1.65
masa added with water at pH
8.5 and 60 F.
7 9 120 units asparaginase/kg of 0.83
masa added with water at pH
8.5 and 60 F.
8 3 120 units asparaginase/kg of 5.32
masa added with water at pH
8.5 and 100 F.
9 6 120 units asparaginase/kg of 4.88
masa added with water at pH
8.5 and 100 F.
9 120 units asparaginase/kg of 4.79
masa added with water at pH
8.5 and 100 F.
11 3 120 units asparaginase/kg of 2.61
masa added with water at pH 6
and ambient temperature.
12 6 120 units asparaginase/kg of 0.87
masa added with water at pH 6
and ambient temperature.
13 9 120 units asparaginase/kg of 0.46
masa added with water at pH 6
and ambient temperature.
TABLE 30: CORN MASA WITH ASPARAGINASE
Table 30 illustrates the effects of pH and temperature on the effectiveness of
asparaginase
addition to corn masa. As shown by comparison of Tests 11-13 with Tests 2-4,
asparagine reduction
is greater at a pH of 6 than a pH of 8.5. Further, while asparaginase was
effective at lower
5 temperatures such as 60 F in reducing asparagine levels as compared to the
control as demonstrated
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by Tests 5-7, the asparagine reduction was more effective at warmer, ambient
temperatures as
demonstrated by Tests 2-4. As indicated by comparing Tests 8-10 with Tests 2-
4, elevating the
temperature to 100 F while the pH is 8.5 does not appear to increase the
reduction of asparagine.
A similar example is shown by Table 31 set out below. First, corn was cooked
to a
moisture level of 45%. This corn is then milled for 1 minute, during which
time the enzyme
asparaginase is added in an aqueous solution by an enzyme addition pump
operated at various
frequencies. As with the previous test, the resultant masa is quenched in
samples taken at 3, 6, and 9
minutes. The level of asparagine found in such samples is then measured. As
shown by comparing
Tests 5-7 with Tests 2-4, the impact of having an elevated temperature may not
be very substantial
for low residence times as indicated by comparing Test 5 and Test 2. However,
at residence times of
6 and 9 minutes, the impact of having an elevated temperature increases the
asparagine reduction in
corn masa. Further, as demonstrated by Tests 8-16, the operation of the enzyme
pump at various
frequencies can have an impact on asparagine reduction.
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Test Run Set Time (mins) Description Asparagine
n-Mole
1 Control 38 5.29
2 3 Mill water 60 F, pH 6, 1980 3.31
units as araginase/k mass
3 6 Mill water 60 F, pH 6, 1980 1.49
units asparaginase/kg masa
4 9 Mill water 60 F, pH 6, 1980 0.71
units as araginase/k masa
3 Mill water 100 F, pH 6, 1980 3.54
units as ara inase/k masa
6 6 Mill water 100 F, pH 6, 1980 1.03
units as ara inase/k masa
7 9 Mill water 100 F, pH 6, 1980 0.30
units as ara inase/k masa
8 3 Mill enzyme pump 10Hz, 1320 4.50
units asaraginase/k masa
9 6 Mill enzyme pump 10Hz, 1320 3.40
units as araginase/k masa
9 Mill enzyme pump 10Hz, 1320 3.30
units asparaginase/kg masa
11 3 Mill enzyme pump 30Hz, 1320 3.11
units as ara inase/k masa
12 6 Mill enzyme pump 30Hz, 1320 1.29
units as araginase/k masa
13 9 Mill enzyme pump 30Hz, 1320 0.73
units as araginase/k masa
14 3 Mill enzyme pump 60Hz, 1320 4.08
units as araginase/k masa
6 Mill enzyme pump 60Hz, 1320 2.01
units asaraginase/k masa
16 9 Mill enzyme pump 60Hz, 1320 0.81
units asparaginase/kg masa
TABLE 31: Corn Masa With Asparaginase
A similar corn chip example is illustrated in Table 32 set out below. In this
test, raw corn is
cooked to a moisture level of 53%. Approximately 30 lbs. of corn is then
spread out on a tray and
sprayed with a water solution containing the enzyme asparaginase. This sprayed
corn is allowed to
5 sit for either 5 or 15 minutes ("sit time") and then milled for one minute.
Samples of the masa are
then taken and quenched at 3, 6, and 9 minutes as previously described. The
level of asparagine is
then measured for each sample.
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Test Run Description Asparagine
n-Mole
1 Control 5.54
2 15,900 units asparaginase, 5 min sit time, 0.68
3 min quench time
3 15,900 units asparaginase, 5 min sit time, 0.37
6 min quench time
4 15,900 units asparaginase, 5 min sit time, 0.41
9 min quench time
15,900 units asparaginase, 15 min sit 0.45
time, 3 min quench time
6 15,900 units asparaginase, 15 min sit 0.35
time, 6 min quench time
7 15,900 units asparaginase, 15 min sit 0.30
time, 9 min quench time
8 80,000 units asparaginase, 5 min sit time, 0.36
3 min quench time
9 80,000 units asparaginase, 5 min sit time, 0.21
6 min quench time
80,000 units asparaginase, 5 min sit time, 0.23
9 min quench time
11 80,000 units asparaginase, 15 min sit 0.53
time, 3 min quench time
12 80,000 units asparaginase, 15 min sit 0.31
time, 6 min quench time
13 80,000 units asparaginase, 15 min sit 0.22
time, 9 min quench time
TABLE 32: Corn Masa With Asparaginase
The tests illustrated by Tables 30, 31, and 32 demonstrate further Applicants'
disclosure that
asparaginase can be used effectively in fabricated foods by addition during
milling or dough
formation or, alternatively, by treating the raw food ingredients prior to
milling or dough formation.
5 XXIV. COMBINATIONS OF ASPARAGINASE AND OTHER ACRYLAMIDE REDUCING AGENTS
In addition to using asparaginase as the sole means of reducing acrylamide in
a thermally
processed food, asparaginase can also be combined with other compounds, such
as divalent and
trivalent cations and various amino acids, for the purpose of reducing the
acrylamide in the final
product. One example of this approach involves the use of a lime soak
comprising calcium
10 hydroxide (divalent cation) of a potato slice combined with a treatment of
the potato slice with an
asparaginase solution.
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In this example, for each test that was run, first 600 grams of potatoes were
peeled and sliced
to a thickness of 0.053 inches. These potato slices were then soaked in 17
liters of water pursuant to
the parameters of each individual test. After the soaking step, the wet slices
are collected and dried
on napkins and then tested for the level of asparagine. In the first test run,
the slices are soaked for
two minutes at 120 F. In the second test run, the slices are soaked for two
minutes at 120 F in the
presence of 1,000 units of asparaginase enzyme. In the third test run, the
slices are soaked for two
minutes at 120 F in a lime solution at a pH of 9. In the fourth test run, the
slices are soaked for two
minutes at 120 F in a lime solution at a pH of 9 in the presence of 100,000
units of the asparaginase
enzyme. The results of this test are reflected in Table 33 set out below.
Test Run Description n-Mole Asparagine
1 Water Soak 681
2 Water w/Asparaginase Soak 480
3 Water with Lime Soak 146
4 Water with Lime and Asparaginase Soak 106
TABLE 33: Effect of Combination of Reducing Agents on Potato Slices
As can be seen from Table 33, the use of either asparaginase or a lime soak
alone will reduce
the amount of asparagine found in the potato slices and, consequently, the
ultimate production of
acrylamide. However, the combination of the use of both asparaginase and lime
in the soak was
even more effective in this regard. Thus, lime can be used to hydrolyze the
cell wall of potato slices
and weaken it sufficiently for an enzyme such as asparaginase to react with
free asparagine or for the
lime to form a complex compound with asparagine. The asparagine level
remaining for production
of acrylamide can be reduced in either situation. Additional data from
experiments using lime is
presented in Table 38 below.
A good effect on reducing acrylamide in thermally processed foods has also
been noted using
the combination of sodium salts, such as sodium phosphate and sodium chloride,
with the amino
acid Lysine. It should also be noted that the use and sequence of any of the
approaches disclosed
individually for reducing acrylamide can yield improved results. For example,
it is possible to treat
a food ingredient with an amino acid followed by treatment with asparaginase,
or vice versa, in
addition to using both agents in combination during one step. Likewise, a food
ingredient can be
treated with a multivalent cation before, after, or in conjunction with
treatment with asparaginase.
Consequently, the formation of acrylamide can be reduced in a thermally
processed food by the use
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of asparaginase in combination with at least one other acrylamide-reducing
agent. Such one other
acrylamide-reducing agent can be selected from the group consisting of free
amino acids, cations
having a valence of at least 2, food grade acids, food grade bases, and a free
thiol compound in
combination with a reducing agent. Such acrylamide-reducing agents can be more
specifically those
agents previously disclosed herein. For example, the amino acid to be used can
be chosen from the
group consisting of cysteine, lysine, glycine, histidine, alanine, methionine,
glutamic acid, aspartic
acid, proline, phenylalanine, valine, arginine, and mixtures thereof.
Consequently, by reference to
the groups of various acrylamide-reducing agents, Applicants intend to
incorporate in this novel
approach all of the individual compounds previously disclosed as being a part
of those groups, any
one of which can be used in combination with asparaginase for the purpose of
reducing acrylamide
formation in thermally processed foods.
XXV. EFFECT OF PH
The example above using a lime soak with a potato slice also demonstrates the
potential
effect of pH on the formation of acrylamide. It has been found that exposing a
food product to either
a high or a low pH solution can ultimately reduce the amount of acrylamide
formation. In addition
to the examples above found in Table 30 and Table 33, in this example, the
reduction of acrylamide
by means of an acetic acid soak is demonstrated. In a first test run, 400
grams of potatoes are peeled
and sliced to .053 inches. These slices are then fried to a moisture level by
weight of 1.1 % and
analyzed for acrylamide. In a second test run, 800 grams of potatoes are
similarly sliced and then
soaked in 4.9 liters of water and 75 milliliters of glacial acetic acid at
room temperature for 60
minutes. These slices are then removed, dried, and fried as with the first
test run. A second test run
involves soaking 800 grams of potato slices in 4.85 liters of water and 150
milliliters of glacial acetic
acid at room temperature for 60 minutes. Thereafter, again the slices are
removed, dried, fried, and
analyzed for acrylamide formation. In a fourth test run, 800 grams of sliced
potatoes are soaked in
4.9 liters of water and 75 milliliters of glacial acetic acid at 120 F for 15
minutes. Thereafter, the
slices are removed, dried, fried, and analyzed. Finally, in a fifth test run,
800 grams of potato slices
are soaked in 4.85 liters of water and 150 milliliters of glacial acetic acid
at 120 F for 60 minutes.
Again, the slices are removed, dried, fried, and analyzed. The results of this
experiment are
demonstrated in Table 34 set out below.
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Test Run Description ASN
Mole
1 Control 203.9
2 4.9 liters water, 75 ml acetic acid, 60 min. 179.0
3 4.85 liters water, 150 ml acetic acid, 60 min. 120.2
4 4.9 liters water, 75 ml acetic acid, 120 F, 15 min. 96.0
4.85 liters water, 150 ml acetic acid, 120 F, 60 min. 62.3
TABLE 34: Effect of Acetic Acid and Asparaginase
Tests 2 and 3 in Table 34 show that more acetic acid results in a greater
reduction in
asparagine with all other factors equal, even at ambient temperatures. Thus,
whereas Table 30
demonstrates a lowered pH can result in a reduced asparagine level in
fabricated food products,
5 Table 34 demonstrates that soaking potato slices in an acidic solution with
a lowered pH can
significantly reduce the level of asparagine, even without the addition of
asparaginase. Further, the
comparison of Tests 3 and 5 reveals that an elevated temperature in the
presence of an acid can
significantly lower the asparagine reduction in potato slices. Further,
comparing Tests 2 and 4
reveals that an elevated temperature can result in a greater reduction of
asparagine, even with a
reduced residence time.
Examples such as those illustrated in Table 33 and Table 34 above demonstrate
that varying
the pH away from neutral can affect the amount of acrylamide produced in a
product that is exposed
to an either acidic or basic solution prior to processing. A similar fact has
been noted when
acrylamide formation is measured when combining asparagine and glucose in a
sodium phosphate
buffer heated at 150 C. The lower the pH of the sodium phosphate buffer, the
less the amount of
acrylamide produced, particularly when the pH is at 5 or below. Similar
results have been noted of
the effect of pH on acrylamide formation in potato flakes when the addition of
calcium chloride,
phosphoric acid, or citric acid is added to reduce the pH of the sample.
Figure 14 graphically illustrates the effect on acrylamide levels of
polyvalent cations which
lower pH. Salt solutions (3 ml) were added to 3 g of potato flakes in a glass
vial. The amount of
calcium chloride was 0.0375 g to 3 g of potato flakes (1.25%). The
concentrations of the calcium
salts and magnesium chloride were adjusted so that the same moles of divalent
cation were added to
the potato flakes. For sodium chloride, the moles of sodium were doubled. The
pH 1404 of the
potato flake slurries were measured before the glass vials were sealed and
heated at 120 C for 40
minutes. Acrylamide 1402 after heating was measured by GC-MS. The control
sample was 3 g of
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potato flakes with 3 ml of deionized water.
As shown by Figure 14, polyvalent cations that lower the pH 1404 of a solution
are
particularly effective at reducing acrylamide 1402. The effect of polyvalent
cations on the pH of a
solution is related to the solubility of the cation/anion pair in the solution
to which the pair is added.
For example, Figure 15 graphically illustrates the effect on pH of calcium
chloride or sodium
chloride to a 0.5 M phosphate and a 0.5 M acetate buffer. Since the alkaline
forms of calcium
phosphate are not soluble, the solution becomes more acidic, as indicated by
the line 1502 that
represents the declining pH as the molar concentration of calcium chloride
increases. Similarly,
when calcium chloride is added to the acetate buffer, the decrease in pH was
smaller as indicated by
line 1504 because the calcium acetate is soluble. When sodium chloride is
added to the acetate
buffer as indicated by line 1506 or to the phosphate buffer as indicated by
line 1508, there was only
small decrease in pH because both sodium acetate and sodium phosphate are
soluble.
Further, the anion portion of the polyvalent cation salt is also a factor that
can affect pH.
Strongly dissociated anions like chlorine have less of an effect on pH than
weakly dissociated anions
like acetate, which can make the pH more alkaline by shifting the reaction
below towards the right.
CH3OOO- + H2O H CH3COOH + OH-
Referring back to Figure 14, Table 35 set out below shows the pKa value of the
salt anion.
Polyvalent Salt Resultant Anion Acid pKa of Anion Acid
Calcium chloride HCl 0.00
Magnesium chloride HCl 0.00
Calcium Gluconate Gluconic 3.60
Calcium Acetate Acetic Acid 4.76
Calcium Citrate Citric Acid 6.39
Table 35: pKa of Anion acids shown in Figure 14.
Based upon the data for calcium salts provided in Figure 14 and Table 35
above, it appears
that higher pKa values for the anion acid appears to make the solution more
alkaline and counteracts
the effect of the calcium on lowering the pH. The salts that most
significantly reduced acrylamide,
calcium chloride, magnesium chloride, and calcium gluconate had anions with
pKa values of less
than 4. The addition of calcium citrate, with an anion pKa value of 6.39
resulted in an acrylamide
level that was higher than the level in potato flakes that occurred with no
added salts, e.g., the level
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revealed in the "water" sample. Consequently, in one embodiment of the present
invention, a pH
lowering salt is used to reduce acrylamide. In one embodiment, the pH lowering
salt comprises a
pKa of less than about 6Ø Such salts include, but are not limited to,
calcium chloride, calcium
lactate, calcium malate, calcium gluconate, calcium phosphate monobasic,
calcium acetate, calcium
lactobionate, calcium propionate, calcium stearoyl lactate, magnesium
chloride, magnesium citrate,
magnesium lactate, magnesium malate, magnesium gluconate, magnesium phosphate,
magnesium
sulfate, aluminum chloride hexahydrate, aluminum chloride, ammonium alum,
potassium alum,
sodium alum, aluminum sulfate, ferric chloride, ferrous gluconate, ferrous
fumarate, ferrous lactate,
ferrous sulfate, cupric chloride, cupric gluconate, cupric sulfate, zinc
gluconate, and zinc sulfate.
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Salt Effective pKa
Calcium Chloride 0.00
Calcium phosphate, monobasic 2.16
Calcium lactate 3.08
Calcium stearoyl lactate 3.08
Calcium gluconate 3.60
Calcium lactobionate 3.60
Calcium acetate 4.76
Calcium propionate 4.86
Calcium malate 5.11
Magnesium chloride 0.00
Magnesium sulfate 1.98
Magnesium chloride 0.00
Magnesium sulfate 1.98
Magnesium phosphate, monobasic 2.16
Magnesium lactate 3.08
Magnesium citrate 3.14
Magnesium malate 3.40
Magnesium gluconate 3.60
Aluminum chloride hexahydrate 0.00
Aluminum chloride 0.00
Ammonium alum 1.98
Potassium alum 1.98
Sodium alum 1.98
Aluminum sulfate 1.98
Ferrous gluconate 3.60
Ferrous fumarate 4.44
Cupric chloride 0.00
Cupric sulfate 1.98
Cupric gluconate 3.60
Zinc sulfate 1.98
Zinc gluconate 3.60
TABLE 36A. EFFECTIVE PKA OF POLYVALENT CATION SALTS.
Different foods require different pH levels during different points in the
process of making
such foods in order to give the foods their unique characteristics. For
example, soft pretzels
generally require a caustic bath in order to taste like a soft pretzel.
Consequently, one skilled in the
art will need to use the various pH levers within the requirements for each of
the foods to be treated.
Consequently, the use of food grade acids and food grade bases, as those terms
are known in the art,
are acrylamide reducing agents.
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XXIV. COMBINATIONS OF ACRYLAMIDE REDUCING AGENTS AND CELLULAR DISRUPTION
The enzyme asparaginase reacts with asparagine and therefore can be utilized
to selectively
remove asparagine, from potatoes. One challenge is to access the asparagine
located inside the cell
wall of a potato without destroying the structural integrity of the tuber.
Consequently, many
embodiments of the present invention are directed towards the weakening of the
cell wall of a plant-
based food comprising asparagine. The cell wall can be weakened, according to
various
embodiments of the present invention, by one or more cell weakening
mechanisms. As used herein,
a "cell weakening mechanism" is defined as any physical or chemical mechanism
that results in
weakened or penetrated cell walls and thereby enhances the ability of an
acrylamide or asparagine
reducing agent to penetrate the cell wall can be used so that, for example,
the enzyme asparaginase
can penetrate the slices, reduce asparagine, and lead to a reduced amount of
acrylamide in a
thermally processed food product. Weakening of the cell wall permits easier
penetration of
asparaginase into the cell so the asparaginase can inactivate asparagine, a
known pre-cursor of
acrylamide. In one embodiment, the weakening of the cell wall occurs at an
elevated temperature of
between about 100 F and about 212 F.
Temperatures in the higher portion of the above range can be used to weaken
the cell walls in
doughs used to make fabricated foods. Temperatures in the lower portion of the
above range, e.g.,
from about 100 F to about 150 F and more preferably 100 F to about 120 F can
be used to weaken
the cell walls of a whole or non-fabricated food such as a sliced potato.
One way to weaken or penetrate the cell wall is to treat potato slices with
the power of
ultrasonic energy to weaken the cell wall and help allow enzyme to penetrate
the interior of the cell
wall. In one embodiment, the ultrasonic energy is applied for at least 30
seconds. In one
embodiment, the ultrasonic energy is applied for between about 30 seconds and
about 60 minutes.
Of course, these ranges are provided for purposes of illustration and not
limitation. Any
synergistically effective amount of ultrasonic energy can be applied to the
food product.
Synergistically effective amounts are amounts that either (a) achieve a
greater percentage
reduction of acrylamide or asparagine than is achieved in a food product using
any type of
acrylamide reducing agent alone; or (b) reduces acrylamide concentration or
asparagine
concentration in a comparable amount to a single acrylamide reducing agent or
asparagine reducing
agent, with fewer the collateral effects on the characteristics (such as
color, taste, and texture) of the
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final product from the addition of an acrylamide or asparagine reducing agent
to the manufacturing
process.
Several tests were conducted to evaluate the relationship of asparagine
reduction in potato
slices treated with ultrasonic energy under various unit operation conditions.
In each ultrasonic test
600 grams of potatoes were peeled and sliced to a thickness of about 0.053
inches and soaked for
about 40 minutes in about 17 liters of water held at about 120 F under four
different test conditions.
Three potato slices from each test were analyzed for asparagine and, for each
test, the average was
reported.
A control sample, Test 1, consisted of placing about 600 grams of peeled
potatoes sliced at
about 0.053 inches in water at about 78 F for about 2 minutes. Three slices
were tested for
asparagine and revealed an average asparagine concentration of about 1.96% by
weight. Unless
otherwise indicated, all units on asparagine concentration is in weight
percent. In Test 2, potato
slices were soaked in water at about 120 F for about 40 minutes and revealed
an asparagine
concentration of about 0.77% by weight, about a 61% reduction over the
control. Test 3 repeated
Test 2 and included about 100,000 units of asparaginase in the water and
revealed an asparagine
concentration of about 0.44% by weight, about a 78% reduction over the
control. Test 4 repeated
Test 3 with ultrasonic energy in an ultrasonic soaker (available from Branson
Ultrasonics Corp of
Danbury, Connecticut) at about 68 kHz applied to the potato slices and
revealed an asparagine
concentration of about 0.10% by weight, about a 95% reduction in asparagine.
Test 5 repeated Test 4
except ultrasonic energy at about 170 kHz instead of about 68 kHz was applied
to the slices and
revealed an average asparagine concentration of about 0.11 % by weight, about
a 94% reduction in
asparagine. The test results are summarized in the Table 36B below.
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Test Time in Solution Ultrasonic Asparagine Reduction
Soak Energy Wt%
1 2 minutes 78 F water -- 1.96 0%
2 40 minutes 120 F water -- 0.77 61%
3 40 minutes 120 F 100,000 -- 0.44 78%
units of
asparaginase
4 40 minutes 120 F 100,000 68 kHz 0.10 95%
units of
asparaginase
40 minutes 120 F 100,000 170 kHz 0.11 94%
units of
asparaginase
TABLE 36B. COMPARATIVE ANALYSIS SONICATION OF POTATO SLICES IN ENZYME SOLUTION
The data in the Table 36B clearly supports the theory that the application of
ultrasonic energy
to a potato slice can further lower the asparagine concentration. Test 4 had a
22% greater reduction
of asparagine ([78%-95%]/78%) than Test 3. As exemplified by Test 2, soaking
in water at an
5 elevated temperature can also make the cell wall more porous.
Regarding physical mechanisms, in one embodiment, the cell wall is weakened by
application of a vacuum to the slices. In one embodiment, slices are treated
with lime and then
soaked into an enzyme solution under vacuum. Without being limited to theory,
it is believed that
the cell wall expands when a vacuum is released and at this point the enzyme
can penetrate the cell
wall. Prior treatment with lime or other intervention such as sonication can
weaken the slices and
under vacuum these treated slices can weaken even more easily.
In one embodiment, a pressure differential is used to force an acrylamide
reducing agent such
as asparaginase into the potatoes. As used herein a pressure differential is
defined as a pressure
different from the atmospheric pressure and the pressure differential can
impart a positive pressure
or a negative pressure (vacuum). For example, potatoes can be exposed to a
vacuum of 20 to 30
psig in the presence of an asparaginase solution or other acrylamide reducing
agent. Higher levels of
vacuum application including a pure vacuum can cause cell walls to burst.
Without being bound to
theory, it is believed that lower levels of vacuum application may not
sufficiently expand the
interstitial spaces within the potato cells to permit an acrylamide reducing
agent to penetrate the
potato slice.
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In one embodiment, the pressure differential comprises a pulsed differential
or cycle of
positive or negative pressure to create and release a vacuum a number of times
so that the cell wall
experiences multiple expansions and contractions to weaken or puncture the
cell surface thereby
improving the chances of enzyme penetration into the cell wall. In one
embodiment, the pressure
differential is applied for at least two cycles.
Several tests were conducted to evaluate the relationship of asparagine
reduction in potato
slices treated with a vacuum under various unit operation conditions. In each
test, 420 grams of
potatoes were peeled and sliced to a thickness of 0.053 inches. Unless noted,
four potato slices from
each test were analyzed for asparagine and the average for each test was
reported. Each test utilized
about 210 grams of potato slices and about 7 liters of water. The tests
occurred in two temperatures
of water, an ambient temperature of about 75 F and an elevated temperature of
about 120 F. The
soak times were varied as was the addition of asparaginase into the solution.
Further, some samples
were placed into a vacuum infusion unit and held at -20 psi. A vacuum infusion
unit that can be
used is a vacuum tumbler model VTS-42 available from Biro Manufacturing
Company of
Marblehead, OR The test conditions and results are summarized in the table
below.
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ASN
(mol) Reduction
Soak
Soak Time Temperature 7,000 Units
Test (min) (F) 20 psi Vac Enzyme average %
1 6 120 704 --
2 6 120 530 25
3 6 120 x 509 28
4 6 120 x x 572 19
3 x 2 120 x 545 23
6 3 x 2 120 x x 439 38
7 6 Ambient 449 36
8 6 Ambient x 153 78
9 6 Ambient x x 168 76
3 x 2 Ambient x 131 81
11 3 x 2 Ambient x x 175 75
12 12 Ambient 138 80
13 12 Ambient x 233 67
14 12 Ambient x x 133 81
6 x 2 Ambient x 107 85
16 6x2 Ambient x x 71 90
* Number average for three tests.
TABLE 37. Effect of the to Evaluate Impact of Vacuum / Pulse Vacuum on Potato
Slices
In Test 1, potato slices were soaked for six minutes at 120 F. In Test 2,
potato slices were
soaked for 6 minutes at 120 F in 14 liters of water having 7000 units of
enzyme. In Test 3, potato
5 slices were soaked for 6 minutes at 120 F in 14 liters of water under a 20
psi vacuum in the vacuum
infuser unit. In Test 4, potato slices were soaked for 6 minutes in 14 liters
of water at 120 F with
7000 units of enzyme under 20 psi of vacuum in the vacuum infuser unit. In
Test 5, potato slices
were soaked for three separate two-minute intervals in 14 liters of water at
120 F under 20 psi of
vacuum. In between each two-minute interval, the vacuum was released and
reapplied. In Test 6,
10 potato slices were soaked for 3 two-minute intervals in 14 liters of water
at 120 F with 7000 units of
enzyme under 20 psi of vacuum. Again, between each interval, the vacuum was
released and
reapplied. In Test 7, potato slices were soaked for 6 minutes at ambient
temperature. In Test 8,
potato slices were soaked for 6 minutes at ambient temperature in 14 liters of
water under a 20 psi
vacuum. In Test 9, potato slices were soaked for 6 minutes in 14 liters of
water at ambient
15 temperature with 7000 units of enzyme under a vacuum of 20 psi. In Test 10,
potato slices were
soaked for 3 two-minute intervals at ambient temperature in 14 liters of water
under a 20 psi
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vacuum. Again, between each interval, the vacuum was released and reapplied.
In Test 11, potato
slices were soaked for three, two-minute intervals in 14 liters of water at
ambient temperature with
7000 units of enzyme under a 20 psi vacuum. Between each interval the vacuum
was released and
reapplied. In Test 12, potato slices were soaked for 12 minutes at ambient
temperature. In Test 13,
potato slices were soaked for 12 minutes at ambient temperature in 14 liters
of water under a vacuum
of 20 psi. In Test 14, potato slices were soaked for 12 minutes in 14 liters
of water at ambient
temperature with 7000 units of enzyme under a vacuum of 20 psi. In Test 15,
potato slices were
soaked for six, two-minute intervals at ambient temperature in 14 liters of
water under a 20 psi
vacuum. In between each interval the vacuum was released and reapplied. In
Test 16, potato slices
were soaked for six, two-minute intervals in 14 liters of water at ambient
temperature with 7000
units of enzyme under at 20 psi vacuum. Again, between each interval the
vacuum was released and
reapplied.
The data in the Table 37 clearly supports the theory that the application of a
vacuum to a
potato slice can further lower the asparagine concentration. For example, Test
3, which used a
vacuum had a 12% greater reduction of asparagine ([25%-28%]/25%) than Test 2.
Similarly, Test 8
had over 100% greater reduction of asparagine than Test 7. This result may be
exaggerated due to
differences in native asparagine levels between the test samples. The potato
used for Test 13, which
had a higher level of asparagine than Test 12 even though Test 13 utilized a
vacuum, most likely had
a much higher level of native asparagine than the potato used in Test 12.
Further, as indicated by Test 6, when the vacuum is applied in a pulsed
manner, or when the
vacuum is released, reapplied and released three different times, the
asparagine reduction shoots up
to 38% from 19% in Test 4 when enzyme is used in the solution. Further, in
comparing Test 16 with
Test 14 use of a pulsed vacuum resulted in more than a 10% greater reduction
in asparagine ([81 %-
90%]/81 %) Thus it is clear that a vacuum can be used in a pulsed manner to
effectively reduce the
amount of asparagine in potato slices.
In one embodiment, the potato slices can be washed with other suitable
chelating agents, or
agents that complex with asparagine such that asparagine is no longer
available for the acrylamide
reaction.
Several tests were run to evaluate the relationship of potato slices treated
with lime under
various unit operation conditions. The results are listed in the Table 38
below.
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ASN
Reduction
Test
No. Test Condition average %
Test 1 2 min/nn Temp 0.93 0
Test 2 6 min/120 F 0.92 1
Test 3 6min/120 F/2% lime 0.51 45
Test 4 6 min/120 F/2% lime/Vacuum/Enzyme 0.63 33
Test 5 6 min/ 120 F/Enz me/Vacuum 0.54 42
Test 6 6 min/120 F/2% lime/Enzyme/Vacuum 0.56 39
6 min/120 F/2%
Test 7 lime/vacuum/Enzyme/Rinsed/Enzyme 0.26 72
TABLE 38. EFFECT OF THE TO EVALUATE IMPACT OF LIME ON POTATO
For each test, 840 grams of potatoes were peeled and sliced at a thickness of
0.053 inches and
soaked in 28 liters of water. In Test 1, potato slices soaked in water for 2
minutes at ambient
temperature. In Test 2, potato slices were soaked for 6 minutes in water at
120 F. Variation in
native levels of asparagine are the likely cause of the similar asparagine
concentrations in Test 1 and
Test 2. In Test 3, potato slices were soaked for 6 minutes in water at 120 F
with a 2% lime solution.
In Test 4, potato slices were soaked for 6 minutes at 120 F in a 2% lime
solution under a 20 psi
vacuum. Slices were then rinsed and soaked for 10 minutes in 28 liters of
water at 120 F with
14,000 units of enzyme. In Test 5, potato slices were soaked for 6 minutes at
120 F in 28 liters of
water having 14,000 units of enzyme under vacuum at 20 psi. In Test 6, potato
slices were soaked
for 6 minutes in 2% lime at 120 F. The potato slices were then rinsed for 5
minutes and then soaked
for 10 minutes in 28 liters of water having 14,000 units of enzyme at 120 F
under a vacuum of 20
psi. In Test 7, potato slices were soaked for 6 minutes at 120 F in a 2% lime
solution under a 20 psi
vacuum. The slices were rinsed for 5 minutes and soaked for 10 minutes in 28
liters of water having
14,000 units of enzyme and at 120 F. As shown by Test 3, soaking in a 2% lime
solution instead of
water alone results in a significantly higher asparagine reduction. The level
of lime disclosed above
is for purposes of illustration and not limitation. In one embodiment, the
slices can be soaked in a
0.1 % to about a 2% by weight lime solution. Lime concentrations higher than
2% by weight can be
used, but such levels may begin to impact finished product flavor.
Another way to penetrate the cell wall is to pre-heat the raw slices via
microwave energy so
that the moisture removed from the interior of the slices (microwave
preferentially removes moisture
from interior of a product rather than its surface) creates pathways or
channels which can be utilized
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for enzyme penetration when the treated slices are soaked in an enzyme
solution. In one
embodiment, a whole potato is microwaved to reduce the internal moisture from
a native about 80%
moisture to about a 60% moisture content. The loss of moisture from within the
potatoes can create
channels which can be utilized for asparaginase to penetrate the interior of
the tuber when the slices
are soaked in an enzyme solution.
Several tests were conducted on potato slices to analyze the additional effect
of microwave
energy on asparagine reduction. In each test, 420 grams of potatoes were
peeled and sliced to a
thickness of 0.053 inches. Unless noted, four potato slices from each test
were analyzed for
asparagine and the average for each test was reported. Each test utilized
about 210 grams of potato
slices soaked in about 7 liters of solution. The tests occurred in two
temperatures of solution, an
ambient temperature of about 75 F and an elevated temperature of about 120 F.
The soak times
were varied as was the addition of asparaginase into the solution. Further,
some samples were placed
into a vacuum infusion unit and held at -20 psi. The test conditions and
results are summarized in
the table below.
ASN
Reduction
Test Condition average %
1* 2 min soak/room temp 1.67 0
2 6 min soak/room temp 0.56 66
3 6 min soak & ASNase &-20 psi vaccum/room temp 0.64 62
4 10 sec. microwave/6 min soak/room temp 0.57 66
5 30 sec. microwave/6 min soak/room temp 0.52 69
6 1 min microwave/6 min soak/room temp 0.54 68
10 sec. microwave/6 min soak & -20 psi & ASNase/room
7 temp 0.53 68
30 sec. microwave/6 min soak & -20 psi & ASNase/room
8 temp 0.53 68
1 min microwave/6 min soak & -20 psi & ASNase/room
9 temp 0.37 78
10 10 sec. microwave/6 min soak & -20 psi & ASNase/120 F 0.56 66
11 30 sec. microwave/6 min soak & -20 psi & ASNase/120 F 0.42 75
12** 1 min microwave/6 min soak & -20 psi & ASNase/120 F 0.50 70
* Number average for three tests.
* * Number from single test.
TABLE 39. Effect of the Revealing Impact of MicrowaveNacuum on Slices
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In Test 1, the control test, potato slices were soaked for 2 minutes at
ambient temperature. In
Test 2, potato slices were soaked for 6 minutes at ambient temperature. In
Test 3, potato slices were
soaked for 6 minutes in 14 liters of water at ambient temperature with 7000
units of enzyme under a
vacuum of 20 psi. In Test 4, potato slices were microwaved for 10 seconds and
then soaked for six
minutes at ambient temperature in 14 liters of water. In Test 5, potato slices
were microwaved for
30 seconds and soaked for 6 minutes at ambient temperature in 14 liters of
water. In Test 6, potato
slices were microwaved for 1 minute and then soaked for 6 minutes at ambient
temperature in 14
liters of water. In Test 7, potato slices were microwaved for 10 seconds and
then soaked for 6
minutes at ambient temperature in 14 liters of water under -20 psi vacuum with
7000 units of
enzyme. In Test 8, potato slices were microwaved for 30 seconds and then
soaked for 6 minutes at
ambient temperature in 14 liters of water under a 20 psi vacuum with 7000
units of enzyme. In Test
9, potato slices were microwaved for 1 minute. The slices were soaked for 6
minutes at ambient
temperature in 14 liters of water under a vacuum of 20 psi with 7000 units of
enzyme. In Test 10,
potato slices were microwaved for 10 seconds. The potato slices were soaked
for 6 minutes at 120 F
in 14 liters of water having 7000 units of enzyme under 20 psi of vacuum. In
Test 11, potato slices
were microwaved for 30 seconds and then soaked for 6 minutes at 120 F in 14
liters having 7000
units of enzyme under 20 psi of vacuum. In Test 12, potato slices were
microwaved for 1 minute
and then soaked for 6 minutes at 120 F in 14 liters having 7000 units of
enzyme under a vacuum of
psi.
20 The use of a microwave can also enhance the reduction of asparagine in
potato slices. For
example, in comparing Test 2 with Tests 4 through 6; with all other factors
being equal, it appears
that pre-treating potato slices in a microwave for 10 seconds has little or no
impact. However, at 30
seconds of microwave pre-treatment, followed by a 6 minute soak at room
temperature, the potato
slices exhibited a 69% reduction in asparagine, which is better than the 66%
reduction achieved with
no microwave pre-treatment.
Pre-treating with a microwave for 1 minute resulted in a 68% reduction of
asparagine.
Additionally, in comparing Test 3 with Tests 7 through 9, the microwave pre-
treatment results in
significantly higher reductions of asparagine. For example, regarding Test 3;
for potato slices that
were soaked for 6 minutes in an asparaginase solution at room temperature
under a vacuum of 20
psi, the slices exhibited a 62% reduction of asparagine. However, when potato
slices were pre-
treated in a microwave for 10 seconds prior to the same treatments of Test 3,
the asparagine
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reduction was 68% and a 1 minute microwave pre-treatment resulted in a 78%
reduction of
asparagine as indicated by Test 9. Consequently, microwave pre-treatment can
facilitate the
reduction of asparagine in potato slices.
In one embodiment, the potato slices are made `leaky' so that large enzyme
molecules such
as asparaginase can penetrate the cell structure and react with the asparagine
in the slice interior.
The pathways can be created either mechanically by docking the surface
(docking see 4,889,733 and
4,889,737) of slices with minute holes with syringes or other mechanical aids.
Alternatively, in one embodiment, the cell weakening mechanism comprises one
or more cell
weakening enzymes. Pathways in the cell wall can be created by means of an
enzyme e.g. cellulase
or hemicellulase that attacks the cell wall of the starch granule. The cell
wall can be weakened by
contacting the cell wall with one or more cell weakening enzymes including,
but not limited to
cellulase, endoglucanase, endo-1,4-beta-glucanase, carboxymethyl cellulose,
endo-1,4-beta-D-
glucanase, beta- 1,4-glucanase, beta-l,4-endoglucan hydrolase,
celludextrinase, avicelase, xylanase,
and hemicellulase. In one embodiment, one or more cell weakening enzymes can
be added together
to make to a cell weakening enzyme solution. The cell weakening enzyme
solution can then contact
a plant-based food to weaken the cell walls of the plant-based food. By
weakening the cell wall with
a cell weakening enzyme, the penetration of asparaginase into the cell wall
becomes easier. Several
tests were conducted on potato slices to analyze the additional effect of an
enzyme that attacks the
cell wall on asparagine reduction. In each test, 840 grams of potatoes were
peeled and sliced to a
thickness of 0.053 inches. Each test utilized about 840 grams of potato slices
soaked about 28 liters
of solution. The tests occurred at an elevated temperature of about 120 F for
a soak time of 10
minutes. The test conditions and results are summarized in the table below.
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ASN
(nmol/g) Reduction
Test Condition average %
1 2 min soak at 120 F 733 0
2 lO min soak at 120 F 493 32.7
3 10 min soak at 120 F pH 4 Citric acid & 5 min rinse 277 62.2
min soak at 120 F @ pH 4 Citric acid/0.84 g
4 VISCOZYME & 5 min rinse 434 40.8
10 min soak at 120 F @ pH 4 Citric acid/0.84 g
VISCOZYME and ultrasonic frequency 68 kHz & 5 min
5 rinse 185 74.7
10 min soak at 120 F @ pH 4 Citric acid/0.84 g
VISCOZYME and ultrasonic frequency 68 kHz & 5 min
6 rinse. 10 min soak 14,000 units enzyme in 28 L of water 33 95.5
TABLE 40. Effect of the Revealing Impact of a Cell Weakening Enzyme on Slices.
In Test 1, the control test, potato slices were soaked in water at 120 F for 2
minutes. After
soaking, the slices were rinsed for 5 minutes and tested for asparagine. In
Test 2, potato slices were
soaked for 10 minutes in water at 120 F. After soaking, the slices were rinsed
for 5 minutes and
5 tested for asparagine. In Test 3, potato slices were soaked for 10 minutes
in 28 liters of water at a
pH of 4 from the addition of citric acid. After soaking, the slices were
rinsed for 5 minutes and
tested for asparagine. In Test 4, potato slices were soaked for 10 minutes in
28 liters of water having
0.84 grams of VISCOZYME at a pH of 4 from the addition of citric acid.
VISCOZYME is an
enzyme cocktail having a range of carbohydrases including arabanase,
cellulase, beta-glucanase,
10 hemicellulase and xylanase. VISCOZYME is available from Novozymes of
Denmark. After
soaking, the slices were rinsed for 5 minutes and tested for asparagine. Test
5 repeated Test 4 with
ultrasonic energy at about 68 kHz applied to the potato slices. Test 6
repeated Test 5 followed by
soaking the potato slices in 28 liters of solution having 14,000 units of
asparaginase for 10 minutes.
The data in the Table 40 clearly supports the theory that the application of a
cell weakening
enzyme in conjunction with asparaginase can substantially reduce the level of
asparagine in a potato
slice. When cell weakening devices are used in conjunction (e.g. ultrasonic
energy simultaneously
with a cell weakening enzyme as shown in Test 5) even greater reductions in
asparagine can occur.
Test 5, for example, had a 20 % greater reduction of asparagine ([62.2%-
74.7%]/62.2%) than Test 3.
As exemplified by Test 6, application of a cell weakening enzyme in
conjunction with ultrasonic
energy can make the cell wall more porous so that asparaginase can effectively
further reduce the
level of remaining asparagine. For example, use of asparaginase after in Test
6 demonstrated a 21 %
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([74.7%-95.5%]/74.7%) greater reduction of asparagine than was achieved in
Test 5 which used no
asparaginase.
In one embodiment, nozzles or probes can be inserted into the potatoes to
`pump' required
amount of asparaginase into the potatoes in a way similar to that utilized to
marinade whole chicken.
While the invention has been particularly shown and described with reference
to several
embodiments, it will be understood by those skilled in the art that various
other approaches to the
reduction of acrylamide in thermally processed foods by use of two or more
acrylamide-reducing
agent additives may be made without departing from the spirit and scope of
this invention. For
example, while the process has been specifically disclosed with regard
primarily to potato and
corn products, the process can also be used in processing of food products
made from barley,
wheat, rye, rice, oats, millet, and other starch-based grains, as well as
other foods containing
asparagine and a reducing sugar, such as sweet potatoes, onion, and other
vegetables. Further,
the process has been demonstrated in potato chips and corn chips, but can be
used in the
processing of many other fabricated food products, such as other types of
snack chips, cereals,
cookies, crackers, hard pretzels, breads and rolls, and the breading for
breaded meats.
Applicants' invention is applicable to all "fabricated snacks," "fabricated
foods," and "thermally
processed foods," as those terms have been defined and explained herein, which
contain
asparagine.
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