Method for retarding staling of baked goods

Intermediate temperature stable bacterial alpha-amylase enzymes having an optimum Phadebas activity above 100% at a temperature of about 65.degree. to 72.degree. C. at a pH of about 5.5 to 6.5 and which retain less than 50% of the Phadebas activity at temperatures above about 75.degree. C., when incorporated in the ingredients used to prepare the baked goods, retard the staling of baked goods without causing gumminess or adversely affecting the organoleptic characteristics of the baked goods. Adding an acid stable alpha-amylase enzyme having an optimum activity at a pH of about 3.0 to 5.0 at a temperature of about 60.degree. to 75.degree. C., to the dough with the intermediate temperature stable alpha-amylase enzyme provides synergistic results in making bakery products by reducing the number of activity units necessary and improving the resistance to staling.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to the use of certain enzyme compositions which can 
be incorporated in the ingredients used to prepare baked goods to improve 
softness and retard staling of the products. 
2. Description of the Prior Art 
The phenomenon of staling of baked goods is not completely understood. 
Staling is usually related to the retrogradation of starch, or the 
association of starch molecules to form areas of crystallinity which 
result in an increase in firmness of the product with the passage of time. 
Staling is of considerable economic importance to wholesale bakeries since 
it limits the shelf life of baked goods in retail outlets to about 3 or 4 
days, plus several additional days in the home of the consumer after 
purchase. The short shelf life of the baked goods has required wholesale 
bakeries to have separate distribution systems that operate independently 
of the usual channels for packaged food distribution. In addition, the 
market area of a bakery is generally limited by the maximum radius the 
distribution system can cover within 24 hours. 
Cereal chemists and bakery technologists have found that various chemical 
emulsifiers have some influence in extending the shelf life of baked 
goods, such as bread. However, chemical emulsifiers are only partially 
effective in reducing bread staling. Monoglycerides and other emulsifiers 
have been added to bread to improve its softness. Although these 
emulsifiers produce a softer bread, they have little influence in reducing 
the rate of bread staling. The term "baked goods" also connotes 
application to such products as rolls, muffins, biscuits, donuts, crackers 
and cake. 
Enzymes derived from bacterial sources have been used or suggested for use 
in baked goods for the specific purpose of inhibiting staling. 
"Heat stable bacterial alpha-amylase" enzyme as the term is used in the 
baking and enzyme industries, most often refers to enzymes made from 
Bacillus subtilis, which are used to inhibit staling. The Bacillus 
subtilis enzyme has a Phadebas activity for baking purposes above 100% at 
temperatures of about 60.degree. to 80.degree. C. at a pH of about 6.2, 
and retains greater than 50% of its Phadebas activity at temperatures 
approaching 90.degree. C. All Phadebas values are expressed relative to 
the value attained at the standard assay temperature of 55.degree. C. This 
value is considered to be 100% activity. This retention of greater than 
50% of its Phadebas activity at temperatures approaching 90.degree. C. 
causes stickiness and gumminess in baked goods which have employed the 
Bacillus subtilis enzyme in the backing process. 
One enzymatic approach to rearding bread staling is disclosed in U.S. Pat. 
No. 2,615,810 to Stone and involves the use of a heat-stable bacterial 
alpha-amylase enzyme to attack gelatinized starch granules during baking. 
A refinement to Stone's approach is described in U.S. Pat. No. 4,299,848 to 
DeStefanis et al which discloses a process for the inactivation of the 
proteolytic enzymes present in commercially available heat stable 
bacterial alpha-amylase enzyme preparations obtained from extracts of 
Bacillus subtilis, Bacillus stearothermophilus or other microbial sources. 
In a further refinement, U.S. Pat. No. 4,654,216 to Carroll et al discloses 
the addition of an enzyme mixture of heat stable bacterial alpha-amylase 
and a pullulanase to dough in proportions of from 0.25 to 5 SKB 
(alpha-amylase units) and 5 to 75 PUN (debranching enzyme units) per 100 
grams of flour. 
G. Bussiere et al in "The Utilization of Alpha-Amylase and Glucoamylase in 
Industrial Baking Technology", Annales De Technologie Agricole, volume 23 
(2) pages 175 to 189 (1974) discloses studies on the role of heat stable 
bacterial alpha-amylases derived from Bacillus subtilis in bread making 
technology. Bussiere et al teaches that heat stable alpha-amylases of 
bacterial origin are effective in retarding staling, but produce a sticky 
bakery product when used at a dosage of 2.5 SKB units or more per 100 
grams of flour. 
A drawback of the Stone, DeStefanis et al, Carroll et al and Bussiere et al 
approaches is the tendency of heat stable bacterial alpha-amylases to 
remain active too long during baking and to cause gumminess in the 
finished product. As a result, these approaches require a degree of 
control over dosages and enzyme ratios which may be impractical to apply 
commercially. 
An alternative to a heat stable bacterial alpha-amylase is described in 
Canadian Patent No. 980,703 to Grampp et al which discloses a thermolabile 
bacterial alpha-amylase without the gumminess causing characteristics of 
conventional bacterial alpha-amylases. However, Grampp et al does not 
disclose staling retardation and staling retardation would not be expected 
with this enzyme because of its thermolability. In terms of stability, the 
enzyme is similar to traditional fungal amylase which is most active at 
temperatures of 50.degree.-55.degree. C. 
A bacterial alpha-amylase that is distinct from the aforementioned heat 
stable bacterial alpha-amylases and thermolabile alpha-amylase is derived 
from Bacillus megaterium available as strain NCIB, No. 11568 deposited in 
the National Collection of Industrial Bacteria, Aberdeen, Scotland. The 
gene coding for this enzyme has been inserted into plasmids. 
Microorganisms containing these plasmids and their use to obtain increased 
yields of the enzyme are disclosed in U.S. Pat. Nos. 4,469,791 and 
4,806,426. This enzyme exhibits intermediate temperature stability in 
relation to the heat stable and thermolabile bacterial alpha-amylases. It 
is described in an article by David et al in Starch/Starke., Vol. 39, No. 
12, pp. 436-440, (1987). However, the use of this enzyme in baked goods 
has not been heretofore disclosed. 
SUMMARY OF THE INVENTION 
The present invention is based upon the discovery that intermediate 
temperature stable bacterial alpha-amylase enzymes retard the staling of 
baked goods without causing gumminess or adversely affecting the 
organoleptic characteristics of the baked goods. More specifically, the 
present invention comprises a process for making baked goods that provide 
resistance to staling by incorporating in the ingredients used to prepare 
the baked goods an intermediate temperature stable alpha-amylase enzyme 
having a maximum Phadebas activity above 100% at a temperature of about 
65.degree. to 72.degree. C. at a pH of about 5.5 to 6.5, and yet which 
retains less than 50% of said Phadebas activity at temperatures above 
about 75.degree. C. 
The use of acid stable alpha-amylase enzyme having an optimum activity at a 
pH of about 3.0 to 5.0 at a temperature of about 60.degree. to 75.degree. 
C., in conjunction with the intermediate temperature stable alpha-amylase 
enzyme provides synergistic results in making bakery products by reducing 
the number of activity units necessary to improve resistance to staling.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
In accordance with the present invention, it has been found that the 
intermediate temperature stable alpha-amylase enzyme retards the staling 
of baked goods without causing gumminess or adversely affecting the other 
organoleptic properties of the baked goods. 
The intermediate temperature stable enzyme used in this invention has a 
maximum Phadebas activity above 100% at a temperature of about 65.degree. 
to 72.degree. C. at a pH of about 5.5 to 6.5, and retains less than 50% of 
its Phadebas activity at temperatures above about 75.degree. C. 
The inventive enzyme survives incorporation in a dough and remains active 
at temperatures above about 60.degree. C. wherein starch gelatinization 
occurs. The enzyme is rapidly inactivated at temperatures above about 
75.degree. C. which occur later during the baking process and thus has no 
tendency to excessively hydrolyze starch and cause gumminess in the 
finished baked goods product. In contrast, the problem of gumminess exists 
with Bacillus subtilis heat stable alpha-amylase because it does not 
inactivate at 75.degree. C. and retains greater than 50% of its Phadebas 
activity at temperatures approaching 90.degree. C. 
Properties of one Bacillus megaterium alpha-amylase enzyme are described in 
the aforementioned article by M. H. David et al in Starch/Starke, Vol. 39, 
No. 12, pp. 436-440, (1987). Use of the enzyme for dextrose production is 
disclosed in U.S. Pat. No. 4,650,757 to M. H. David et al and reported by 
R. E. Hebeda et al in Starch/Starke, Vol. 40, No. 1, pp. 33-36, (1988). 
Activity of the Bacillus megaterium alpha-amylase is determined by a 
Phadebas Dye Release Assay (Pharmacia Diagnostic AB, Uppsala Sweden) as 
follows. An aqueous solution of the intermediate temperature stable 
bacterial alpha-amylase is prepared containing an estimated 0.15-0.6 U/ml. 
Four ml of buffer (0.02M, pH 5.0 acetate buffer in 0.002M CaCl) is added 
into a conical cetrifuge tube along with one Phadebas Amylase Test tablet 
(Pharmacia Diagnostic AB, Uppsala Sweden) and incubated at 55.degree. C. 
for 5 minutes, resulting in a reaction pH of about 6.2. The enzyme 
solution (0.2 ml) is added to the buffer solution and incubation continued 
at 55.degree. C. After exactly 15 minutes, 1.0 ml of 0.5M NaOH is added. 
The reaction mixture is agitated, cooled to room temperature, and 
centrifuged for ten minutes at 1500 G where G is the force of gravity. 
Absorbance of the supernate is measured at 620 nm. A blank is carried 
through in the same manner using water in place of enzyme sample. Units of 
activity (U) are calculated as follows: 
EQU U/g or U/ml=(Abs. of sample-Abs. of blank).times.calibration 
factor.times.dilution factor 
The calibration factor is supplied with the test tablets. 
In practicing this invention, the intermediate temperature stable bacterial 
alpha-amylase enzyme is used at a level of from about 1.0 to about 20, 
preferably about 2.0 to about 10 and most preferably about 3 to about 5 
alpha-amylase units per gram of flour. 
The alpha-amylase enzyme preparation can be employed as a concentrated 
aqueous solution or as a solid. In the baking process, the enzyme can be 
added to either the flour or water during the mixing operation. 
In a further embodiment of the invention, it has been found that the 
combination of an intermediate temperature stable bacterial alpha-amylase 
derived from Bacillus megaterium with an acid stable alpha-amylase derived 
from Aspergillus niger provides synergistic results in retarding the 
staling of baked goods, with less activity units being necessary and 
greater retarding of staling being achieved. An acid stable alpha-amylase 
is described in copending application Ser. No. 419,980, filed Oct. 11, 
1989, a continuation of Ser. No. 166,926, filed Mar. 11, 1988 now 
abandoned. 
For example, in the combined use of Bacillus megaterium intermediate 
temperature stable alpha-amylase with Aspergillus niger acid stable 
alpha-amylase to retard staling, the Bacillus megaterium intermediate 
temperature stable alpha-amylase can be used at a level from about 0.5 to 
10, preferably about 1 to 7, and most preferably about 2 to 4 
alpha-amylase units per gram of flour in combination with the Aspergillus 
niger acid stable alpha-amylase at a level of from about 0.1 to 5, 
preferably about 0.5 to 3, and most preferably about 1 to 2 alpha-amylase 
units per gram of flour. 
Baked goods prepared in accordance with the process of this invention show 
improved antistaling properties and remain softer longer as measured by 
instruments typically used to determine bread softness, such as a Voland 
Pentrometer or Instron Texture Analyzing Apparatus. Typical improvement in 
softness is about 10 to 50% after about 1 to 5 days storage. An additional 
benefit of the enzyme is increased bread loaf volume on the order of about 
3 to 5%. The following examples illustrate specific embodiments of the 
present invention. In the examples and throughout the specification, all 
parts and percentages are by weight, unless otherwise indicated. 
EXAMPLE 1 
A plant scale baking trial was conducted using a plain white bread formula 
and a straight dough process. 
Dough batches of 500 pounds were prepared using unmalted flour in the 
following basic white bread formula: 
______________________________________ 
Ingredients Weight Percent 
______________________________________ 
Flour 61.22 
Water 34.07 
Yeast 2.10 
Sugar 1.70 
Salt 0.91 
100.00 TOTAL 
______________________________________ 
Megafresh.TM. baking carbohydrase available from Enzyme Bio-Systems Ltd., 
Englewood Cliffs, N.J., an intermediate temperature stable bacterial 
alpha-amylase derived from Bacillus megaterium and having a maximum 
Phadebas activity above 100% at a pH of about 5.5 to 6.5 at a temperature 
of about 65.degree. to 72.degree. C., was added at a dosage of 10.9 U/g 
flour to determine the effect of enzyme on the staling properties of the 
bread produced. The enzyme was added to 20 pounds of the formula water, 
mixed, and the solution added to the remainder of the ingredients. As a 
control, 0.05% barley malt, based on flour, was used in place of the 
Megafresh.TM.. 
In each case, the dough was held for 5 minutes floor time before being 
deposited into the hopper of the divider. The dough was then divided, 
intermediately proofed, sheeted and molded, panned and rolled on racks 
into a proof box. After the appropriate amount of time, the racks were 
removed from the proofer and unloaded onto the oven feed conveyor system. 
The pans traveled through the oven, depanner, cooler, slicer and bagger. 
Samples for shelf life studies were stored at room temperature. 
Freshness tests were determined by means of a Voland Penetrometer (Voland 
Company, Hawthorne, N.Y.), having a 1 inch flat probe (Voland 
designation-TA 11). Each of the loaves was sliced into uniform segments. 
Each segment was placed in the penetrometer set for 5 millimeter 
penetration at a speed of 2 millimeters per second. The procedure was 
consistent with that disclosed in an article by Baker et al "Comparison of 
Bread Firmness Measurements by Four Instruments" in CEREAL FOODS WORLD, 
pages 486 to 489, vol. 32, No. 7, (July 1987). The test results for each 
of the slices of bread produced from the various doughs are given in Table 
1 which follows: 
TABLE 1 
______________________________________ 
Voland Penetrometer Load (Grams) (a) 
Storage Control Megafresh .TM. 
Reduction in 
Time, Days 
(Barley Malt) 
10.9 U/g Flour 
Firmness, % 
______________________________________ 
1 258 .+-. 37 155 .+-. 47 40 
2 331 .+-. 121 
253 .+-. 142 
24 
3 342 .+-. 124 
280 .+-. 75 18 
4 510 .+-. 106 
339 .+-. 74 34 
5 610 .+-. 96 298 .+-. 85 51 
______________________________________ 
(a): Average of 15 determinations. 
As can be seen from the data which has been plotted in the graph on FIG. 1, 
firmness of the bread prepared with Megafresh.TM. is reduced by as much as 
51% when compared with the barley malt control. 
EXAMPLE 2 
The procedure of Example 1 was repeated using a combination of 
Megafresh.TM. baking carbohydrase, an intermediate temperature stable 
bacterial alpha-amylase derived from Bacillus megaterium and having a 
maximum Phadebas activity above 100% at a pH of about 5.5 to 6.5 at a 
temperature of about 65.degree. to 72.degree. C., and Multifresh.TM. 
baking carbohydrase, commercially available from Enzyme Bio-System Ltd., 
Englewood Cliffs, N.J., an acid stable alpha-amylase derived from 
Aspergillus niger having an optimum activity at a pH of about 3.0 to 5.0 
at a temperature of about 60.degree. to 75.degree. C. A Megafresh.TM. 
dosage of 4.8 U/g of flour was used in combination with 2.7 U/g flour 
Multifresh.TM.. 
TABLE 2 
______________________________________ 
Voland Penetrometer Load (Grams) (a) 
Storage Megafresh .TM. 
Time, Control Multifresh .TM. 
Reduction in 
Days (Malt) Combination 
Firmness, % 
______________________________________ 
1 258 .+-. 37 100 .+-. 22 
61 
2 331 .+-. 121 
186 .+-. 47 
44 
3 343 .+-. 124 
177 .+-. 65 
48 
4 510 .+-. 106 
183 .+-. 61 
64 
5 610 .+-. 96 188 .+-. 68 
69 
______________________________________ 
(a): Average of 15 determinations. 
The combination of enzymes reduced bread firmness by as much as 69% 
compared to the control after 5 days storage. 
EXAMPLE 3 
A laboratory trial was conducted using a white pan bread formula and a 
sponge-dough process. The mixer was a Hobart A-200 Mixer with 20 quart 
bowl and dough hook. The formula and processing parameters were as 
follows: 
______________________________________ 
White Pan Bread Control Formula 
______________________________________ 
Ingredients Grams 
______________________________________ 
SPONGE: 
Bread Flour 2100 
Mineral Yeast Food 6 
Compressed Yeast 60 
Water 1260 
DOUGH: 
Bread Flour 900 
Granulated Sugar 180 
Nonfat Dry Milk 60 
Salt 60 
All-Purpose Shortening 90 
Crumb Softener GMS-90 30 
Water and Ice (Variable) 570 
Total Weight (Yields 9-10 Loaves) 
5316 
______________________________________ 
Processing Conditions 
SPONGE DOUGH 
______________________________________ 
Desired Temperature: 
76.degree.-77.degree. F. 
78.degree.-80.degree. F. 
Fermentation Time: 
3.25 Hours 10 Minutes 
Scaling Weight: 
526 grams Dough per loaf 
Proof: To Average Total Height of 
100 .+-. 1 mm 
Bake: 16 Minutes at 450.degree. F. 
Cool: One Hour at Ambient Temp- 
erature 
______________________________________ 
Three tests were made using 2.6, 5.1, or 10 U/g flour Megafresh.TM.. In 
each case, the enzyme was added to a portion of the water used for the 
dough, mixed and added to the remainder of the ingredients. The dough was 
processed and bread loaves prepared as above. A control test was made 
without the addition of Megafresh.TM.. Each loaf was packaged in two 
polyethylene bread bags and stored in an environmental cabinet at 
77.degree. F. (25.degree. C.) until withdrawn for evaluation after 3, 7, 
and 11 days. At each time, 3 loaves from each test were evaluated in 
replicate (5 measurements per loaf) for crumb firmness using the Instron 
Texture Analyzing Apparatus by the method described by Baker and Ponte in 
Cereal Foods World, Vol. 32, No. 7, pp 491-493, (July, 1987). Results of 
these tests are given in Table 3 as follows: 
TABLE 3 
______________________________________ 
Instron Texture Analyzing Apparatus (grams) (a) 
Storage Megafresh .TM., U/g Flour 
Time, Days 
Control 2.6 5.1 10 
______________________________________ 
3 219 .+-. 3 
208 .+-. 3 
179 .+-. 2 
188 .+-. 3 
7 320 .+-. 4 
277 .+-. 2 
256 .+-. 5 
260 .+-. 3 
11 403 .+-. 8 
361 .+-. 5 
331 .+-. 6 
346 .+-. 8 
______________________________________ 
(a): Average of 15 determinations. 
The addition of Megafresh.TM. to the dough resulted in improved freshness 
compared to the control. For instance, with the intermediate dosage of 
Megafresh.TM. (5.1 U/g flour), firmness after 3, 7, and 11 days was 18-20% 
less than the control values. 
EXAMPLE 4 
A laboratory trial was conducted in the same manner as described in Example 
3. The formula and processing conditions were as follows: 
______________________________________ 
White Pan Bread Control Formula 
______________________________________ 
Ingredients Grams 
______________________________________ 
SPONGE: 
Bread Flour 2100 
Mineral Yeast Food, Bromated 
3 
Sodium Stearoyl Lactylate 11.2 
Compressed Yeast 75 
Water 1260 
DOUGH: 
Bread Flour 900 
Nonfat Dry Milk 60 
Salt 60 
Calcium Proplonate 3 
Crumb Softener GMS-90 30 
Soybean Oil 60 
42% High Fructose Corn Syrup 
255 
Water and Ice 526 
Total Weight (Yields 9-10 Loaves) 
5343.2 
______________________________________ 
Processing Conditions 
SPONGE DOUGH 
______________________________________ 
Desired Temperature: 
76.degree. F. 78 .+-. 1.degree. F. 
Fermentation Time: 
3.25 Hours 10 Minutes 
Scaling Weight: 
526 grams Dough per loaf 
Proof: To Average Total Height of 
100 .+-. 1 mm. 
Bake: 18 Minutes at 435.degree. F. 
Cool: One Hour at Ambient Temp- 
erature 
______________________________________ 
Three tests were made using 1.3, 2.6, or 5.1 Megafresh.TM. U/g total flour. 
In each case, the enzyme was added to a portion of the water used for the 
sponge, mixed and added t the remainder of the ingredients. The sponge was 
prepared, the dough processed and bread loaves prepared as above. A 
control test was made without the addition of Megafresh.TM.. Each loaf was 
tested as described in Example 3. Results of these tests are given in 
Table 4 as follows: 
TABLE 4 
______________________________________ 
Instron Texture Analyzing Apparatus (grams) (a) 
Storage Megafresh .TM., U/g Flour 
Time, Days 
Control 1.3 2.6 5.1 
______________________________________ 
1 134 .+-. 3 
113 .+-. 2 
110 .+-. 2 
115 .+-. 2 
4 201 .+-. 5 
174 .+-. 4 
178 .+-. 3 
183 .+-. 3 
7 249 .+-. 5 
216 .+-. 4 
209 .+-. 11 
215 .+-. 3 
______________________________________ 
(a): Average of 15 determinations. 
The addition of Megafresh.TM. to the sponge resulted in improved freshness 
compared to the control. For instance, with Megafresh.TM. dosages of 1.3, 
2.6 and 5.1 U/g flour, bread firmness after 4 days was 13, 11 and 9%, 
respectively, less than the control value. 
An additional test was made using a Megafresh.TM. dosage 0.6 U/g of flour 
in combination with 0.3 U/g of flour of Multifresh.TM.. The bread firmness 
after 4 and 7 days was 16% and 20%, respectively, less than the control 
value. 
These tests show that when intermediate temperature stable bacterial 
alpha-amylase is incorporated in the sponge in a sponge-dough process, it 
retards bread staling. They also show that this retarding of bread staling 
can be accomplished by very small amounts of the intermediate temperature 
stable bacterial alpha-amylase enzyme when it is used in combination with 
an acid stable microbial alpha-amylase enzyme. 
EXAMPLE 5 
A comparison of Phadebas activity of Bacillus megaterium intermediate 
temperature stable alpha-amylase and Bacillus subtilis heat stable 
alpha-amylase at conditions of 6.2 pH, varied temperature and 15 minutes 
reaction time provided the following results tabulated as follows and 
plotted in FIG. 2. Phadebas activity determined at the standard assay 
temperature of 55.degree. C. was expressed as 100% activity. Values at 
other temperatures were expressed relative to the value determined at 
55.degree. C. 
______________________________________ 
PHADEBAS % ACTIVITY 
.degree.C. 
Bacillus meaterium 
Bacillus subtilis 
______________________________________ 
55 100 100 
60 103 106 
65 119 118 
70 118 127 
75 58 131 
80 30 101 
85 7 72 
______________________________________