CA1146800A - Enzymatic treatment of wheat flour - Google Patents
Enzymatic treatment of wheat flourInfo
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- CA1146800A CA1146800A CA000345671A CA345671A CA1146800A CA 1146800 A CA1146800 A CA 1146800A CA 000345671 A CA000345671 A CA 000345671A CA 345671 A CA345671 A CA 345671A CA 1146800 A CA1146800 A CA 1146800A
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- gluten
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
- A23J1/12—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from cereals, wheat, bran, or molasses
- A23J1/125—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from cereals, wheat, bran, or molasses by treatment involving enzymes or microorganisms
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
ABSTRACT OF THE DISCLOSURE
The present invention provides a process comprising treating an aqueous slurry or suspension of wheat flour, having a flour content of less than 35% by weight, at an elevated of less than about sixty (60) minutes with an .alpha.-amylase enzyme substantially free of protease activity: the starch component is solubilized and the solid gluten is removed from the liquid phase. The process is effected with a minimum amount of water and indeed, all the water utilized, as well as the solubles and other minor components, end up in the starch fraction, i.e. the process may operate on a pollution-free basis.
The present invention provides a process comprising treating an aqueous slurry or suspension of wheat flour, having a flour content of less than 35% by weight, at an elevated of less than about sixty (60) minutes with an .alpha.-amylase enzyme substantially free of protease activity: the starch component is solubilized and the solid gluten is removed from the liquid phase. The process is effected with a minimum amount of water and indeed, all the water utilized, as well as the solubles and other minor components, end up in the starch fraction, i.e. the process may operate on a pollution-free basis.
Description
:~468~)0 The present invention relates ta a process for treating wheat flour and in particular such a process whereby gluten and starch syrup products are obtained.
Backqround of Invention Wheat flour comprises from about 70-85% starch, from about 9-16% proteinaceous material ~about 12 to 21% gluten), and a number of minor components such as hemicelluloses (pentosans) and ash. The gluten and starch, and starch derived products, especially syrups, are standard commercial items and are generally obtained from wheat flour by a variety of water washing procedures such as that described in Canadian Patent No. 1,037,474. The various processes are actually relatively inefficient solid:solid separations in that both gluten and starch are essentially insoluble in water and consequently, following formation of a wheat flour dough or batter, the solid starch is simply washed from the solid gluten which, as obtained under the processing conditions is in a rubbery/elastic condition in the form of lumps or the like. These current commercial proces3es require a large amount of water for batter preparation and an additional large quantity of water for the actual washing of the starch from the gluten. During the process, some of the soluble flour components, such as the pentosans, simple sugars, soluble proteins, as well as some of the starch, which is solubilized by indigenous enzymes (such as ~-amylase), dissolve in the water. However, such potentially u~eful compo-nents are then lost since they remain in the waste~water. This soluble fraction generally represents from 6 to 10% of the flour substance ahd hence is a significant loss.
Moreover, huge volumes of water are used in the process and consequently the waste waterS contain relatively small amounts (about 1~ ) of the soluble components. If the waste water is merely disposed of, i.e. run to sewer, it becomes a major source of water pollution and, in fact, public authorities are now frowning upon, if not banning, such practices. An alterna-tive is to dry, or at least increase the solids content of, this waste material, a very expensive proposition in view of the large energy requirements. A further complication is that many possible profitable uses for the solubles obtainable upon drying or concentrating are precluded because, in many instances, 114~i8~)0 the solubles are contaminated with a relatively large amount of processing aids, especially sodium chloride. ~s is well known, gluten is by far the most phys~cally functional non-animal protein and is highly valued on account of that characteristic.
However, that functionality or vitality is lost i.e. the gluten becomes denatured, in many ways and in particular by heat, and moreover starch gelatinizes in the presence of heat. Consequently, the varlous wheat flour washing processes are carried out at relatlvely low temperatures, generally about 30C, but in any event ~ignificantly below 60C which is widely considered to be the maximum to which gluten may be subjected during such pro-cessing if its desirable functional properties are to be retained.
Moreover, that temperature is also critical from the viewpoint of the starch in that the latter begins to gelatinize at about 55C. As a result, it would prove virtually impossible to separate a gelatinized starch component from the gluten component since the starch would gel thereby making it unsusceptible to being washed from the gluten.
As stated above, the gluten and starch have many tradi-tional uses, and historically many of these require that the pro-ducts be produced to exacting specifications. For example, gluten is generally produced as vital wheat gluten having a protein con-tent greater than 75% and usually in excess of 80% and in as functional a form as possible. As for starch in general, this is finding new markets as the starting material for the production of various syrups which are being used to an ever increasing extent in, for example, sugar replacement, baking and brewing areas. Purification of syrups is quite difficult and expensive but for many applications pure, for example clear and non-coloured, syrups are required. Consequently, since starting with a purer starch would produce a more pure syrup requiring a less extensive purification, commercially available starch as used to produce syrups is utilized in a pure state to produce the resulting commercially available syrups. In summary, the vast majority of commercially available gluten and starch syrups are produced to quite exacting specifications.
However, in many applications, such as brewing (where the present applicant is a large user of wheat starch and syrup derived therefrom),such exacting standards of quality are, in fact, not required butsuch products are used because, in many 6~0 instances, they are the only available suitable products. This results in increased costs to the user and, when the wider implications are considered, a waste of resources in the form of the energy, etc. required to purify the products, where purification is actually n~necessary~
Processes are known for enzymatically solubilizing and converting starch tovarious products. For example, United States Patent No. 2,583,451 teaches a process for producing dextrose comprising treating pure starch with ~-amylase under non-gelatinizing conditions at temperatures less than about 45C
and for periods in excess of forty-eight hours. United States Patent No. 3,922,201 teaches a similar process where granular starch is treated with a-amylase and glucose isomerase to produce levulose at a temperature below ~hich rapid gelatinization o~
the starch would take place. The examples teach process times of twenty-four hours. There are other known processes of a similar nature, but the process conditions and reactants presently utilized are such that if proteinaceous components, and in particular gluten,were present, then such would be significantly adver~ely affected; for example, in the case of wheat flour, gluten would be denatured and rendered useless, for example, for baking purposes; and indeed, might be solubilized to a lessor or greater extent and cause significant contamination of the syrup and derivatives thereof and render the products unusable.
An object of the present invention is to provide a process which, starting with readily available wheat flour, produce~ a wheat starch syrup and essentially vital wheat gluten rapidly and relatively inexpenslvely.
A further object is to provide such a process wherein substantially all of the wheat flour components are recovered and are incorporated into the process products.
Yet a further objective is to provide such a process in which waste waters, generally a major polluting factor, are totally eliminated while at the same time improving syrup yield.
68~)0 General Statement of Invention It has now been found that wheat gluten, either per se, or when present as the protein component of wheat flour, is not as susceptible to being adversely affected ~y heat as is presently widely believed in this art.
In more detail, it has been found that the starch fraction of wheat flour can be liquefied in the presence of the gluten com-ponent by treatment in aqueous suspension with certain a-amylase enzymes at significantly elevated temperatures in excess of about 65C for a period of not more than about sixty (60) minutes without functional denaturation of the gluten component occurring.
Thus, the original solid:solid system is thereby converted to a liquid:solid system which can readily be subjected to conven-tional separation techniques to provide the desired syrup and gluten products.
Detailed Statement of Invention According to the present invention there is provided a process for the production of ~yrup and vital gluten from wheat flour the process including the steps of:
forming a slurry comprising wheat flour, water and an ~-amylase enzyme which has substantially no protease activity, the slurry having a pH compatible wlth the activity of the specific enzyme employed tgenerallY 5 to 71 and contalning less than about 35%
by welght o~ wheat flour;
maintaining said slurry at a temperature of from 65C to about 95C with agitatlon for a period less than sixty (60)minutes but sufficient to liquefy 9ubstantially all of the starch component of the flour without signiflcantly adversely affecting the gluten component; and subsequently separating the solid gluten from the liquefied starch syrup.
The process proceeds as follows: the starch gelatinizes whereupon the ~-amylase commences to act thereon, breaking the starch polymer down into smaller chain units thus causing liquefac-tion which prevents the viscosity of the reaction mass from in-creasing excessively and the reaction mixture from gelling. The process is completed when the starch has been liquefied to such an extent that the gluten can readily be removed from the resulting syrup. In practice this is when the majority of 1 ~4~ )0 ~ 5 -the starch has been solubilized.
The rate of ltquefaction of the starch increases as the temperature increases. A major limiting factor is the properti-es of the specific ~-amylase enzyme chosen and in particular the temperature range, etc. at which lt operates most effecitvely and, of course, the temperature at which it would be heat-deactivated. Temperatures within the range of about 70C to about 95C and especlally between 80C to about 90C are preferred.
Although, the reaction mass does not significantly gel, the vlscosity does increase to an extent where the efficiency of the realitvely small amount of the enzyme in converting the starch may be significantly impaired. Agitation of the reaction mass is therefore effected. However, such agita-tion must be controlled since a high level thereof has been found to reduce the yield and quality, especially clarity, of the syrup.
To some extent, therefore, the degree/amount of agitation used depends also on the specific quality of syrup or gluten product required.
Allied to the above i9 the concentration of substrate flour in the reaction mass. It has been found that if the concen-tration of flour exceeds about 35% by weight, then the starch component is not sufficiently liquefled to be practically separated.
On the other hand, too dilute a slurry would increase the liqui-factlon time; reduce the capacity of the equipment or necessitate increasing the size of the equipment; increase handling and energy costs,etc. Also a major factor is that the present process lends itself to producing a solubilized starch product, which may be a ~yrup, and which incorporates substantially all the water (and solubles) and is useable, as ls, since the concentration of available carbohydrates is at commercially acceptable levels for many uses without evaporative at other concentration stages being necessary. Use of excessive amounts of water, i.e. very low flour concentrates, would result in the resulting syrup being of low carbohydrate concentration which would reduce its utility in some areas. Consequently, it is preferred that the starting flour slurry contain from about 15% to less than 30% flour. It should be em-phasized that the starting flour material is just that, namely wheat flour as obtained directly from the milling of wheat with no up-grading whatsoever being necessary. Consequently, it is readily ~146~
available and at reasonable cost. It is possible to process flour fractions upgraded in protein but these do not process as well and are obviously more expensive.
The pH of the reaction mass may vary within the quite wide limits and is essentially the pH at which the selected enzyme operates most effectively. However, it has been found that many suitable a-amylase enzymes operate optimally within the range of 6 - 7, and most wheat flour slurries~suspensions have a natural pH of between pH 6 to 7. Consequently, the reaction mass preferably has a pH of from 6 to 7.
The a-amylase enzyme used mNst be substantially free of protease activity and, obviously, have the ability to function at relatively elevated temperatures, in the presence of a ~tabilizing agent (such as calcium chloride and sodium chloride~
if necessary,albeit for only a relatively short period of time.
Moreover, a-amylases which are most active in the pH range of from 6 to 7 are preferred, sinc~ as stated, aqueous slurries of wheat flour have a natural p~ within that range and, consequently, no ad~ustment of the slurry pH is required.
Examples of such a-amylases include certain species of the Bacillus microorganisms such as 8acillus subtilis and Bacillus licheniformis. Specific examples of such enzymes are as follows: f~ 1~
æ (i~ Calbiochem a-amylase - supplied by Calbiochem Co San Diego, California;
(ii) Sigma a-amylase - a 4x crystallized enzyme supplied by S~gma Chemical Co., St. Louis, Missouri;
(iii) Ban1120L - suppl~ed by Novo Industries of Copenhagen, Denmark;
(iv) Tenase - available from Miles Laboratories Inc., Elkhart Indiana;
(v) HT-1000 - available from Miles Laboratories, Inc., Elkhart,~Indiana;
(vi) Termamyl 60L - also supplied by Novo Industries; and (vii) Takatherm1~ also available from Miles Laboratories Inc., Elkhart, Indiana.
ti) to (v) belng derived from ~. subtil~s and (vi) to ~vit being derived ~rom 3 licheniformis.
68~
The concentration of a-amylase enzyme required is relatively small and obviously depends on many factors, especially the activity thereof and the process temperature. As the specific experiments detailed later indicate, there appears to be a minimum concentration of enzyme if complete liquifaction is to be achieved and that minimum can easily be found via simple experiments as described herein. For example, the following concentrations have been found satisfactory.
(a) Ban 12~L - 1 ml to 2 ml /100 g flour, activity being 120 KN~g;
(b) Calbiochem - > 0.05~ by weight, activity being 1477 AU/g;
(c) Termamyl 60L - > O.5% by weight, activity being 60 KNU/g Of course, an amount of enzy;me of suitable activity must be used; in other words the activity and amount of enzyme must be suf~icient to effect the necessary solubiliation within the stated process parameters. That amount of enzyme is termed "the effective amountnand is readily determined for each sel~cted enzyme using the general lnformation given herein.
In the accompan~tng F~gures:
F~g. l is a curve of conversion t~me ~ersus temperature for Termamyl a-amylase;
Fig. 2 is an activity curve obtained using Calbiochem a-amylase and various pH values;
Fig. 3 is a flow sheet wlth mass balance of an embodiment of the invention using Calbiochem ~-amylase;
Fig. 4 is similar to Fig. 3 but wherein agitation of the gluten product is carried out; and Fig. 5 is a similar curve to Fig. 1 but wherein the enzyme is Sigma a-amylase.
:~468~0 The present invention will be further described withreference to, but not limited by, the following specific experiment:
Experimentation The process of the present invention as described in B the following Examples was carried out in a CHAIZ (Nancy, France) apparatus, orig~nally intended as a brewery laboratory mashing un~t. The apparatusincorporates a temperature controlled bath in whlch reactlon cups are lodged and allows for agitation of the reactants via a stirring mechanism which, during all the Examples, was set at 200 r.p.m.
Separation of the s~rup and gluten was effected by centrifugation using a SORVAL RC-3 HGA swinging buc~et head centrifuge. However, other conventional separation techniques such as filtration and even sedimentation can be used, the choice depending to some extent on the required degree of separation of the li~uid from the solid phase and the specific constitution of the liquefied starch and gluten product(s) required.
Procedure A predetermined quantity of flour (usuallylOOg) was mixed with water, (in the ratio of 1:4 unless otherwise stated) and the selected enzyme~ ~ where required in combination with an accompanying stabilizing mixture of calcium chloride and sodium chloride) was added. The resultlng suspension was plac~din the CHAIZ, where the temperature of the bath had been adjusted to the desired value and the stirring immediately started.
The mixture was held, with stirring, at the set temperature for the duration of the process, following which, any further reaction was arrested by rapid cooling. The mixture was then centrifuged to obtain the heavier gluten and as the top phase, the liquefied starch.
In an alternative embodiment, the gluten was re-washed and centrifuged again, the second liquid phase (comprising a very dilute liquefied starch being used as the flour slurry ma~e-up water.
The experimental w~rk carried out to delineate the effects of temperature on the process of the present invention showed that the process was ameniable to being effected over the range of from 70 to up to about 95C, the results at higher temperatures, whilst showing greater conversion efficiency in terms of ~46800 _ g reduced processing times, also indicated that the liquefied starch and gluten products were not signficantly altered from the products at the lower temperatures. Since the available equtpment was most conveniently operated at temperatures of about 70C to 80C with little effect on the experimental time required, evaluations of other process parameters such as enzyme concentration, etc.
were in many instances carried out at temperatures within that range.
In addition, standard operation of the CHAIZ unit allowed only for batch-type processes and consequently, much of the w~rk effected was of that type. However, all indications are that no problems of substance would be encountered on carrying out the process of the present invention on a continuous basis.
Indeed, from a commercial viewpoint, that would be the pr~ferred mode of operation and the design of suitable e~uipment would present little difficulty.
Process parameters which are important to the efficient carrying out of the proces~ of the present invention include:
(a) temperature ~b) enzyme concentration (c) flour (substrate) concentrate (d) pH
(e) flour type Each specific enzyme i9 unique to some extent as regards degree of activity, optimal processing conditions of pH, temperature, etc., and consequently, in practice the actual process, in terms of the ~pecific values of each parameter, will be tailored to suit the specific enzyme. The experimentation work detailed herein does not, obviously, provide such data for each and every possible ~-amylase. However, the extensive data is fully sufficient for a man skilled in the art to determine, with a few simple experiments, the processing conditions to be most advantageously used with any selected enzyme.
~6800 Effect o~ ~eat, per se, on ~ital ~heat ~-luten In this experiment the effect of heat, per se, on vital gluten was determined by reconstituting standard regular vital wheat gluten (obtained from IGP Limited, Montreal) with distilled water and, following reconstitution, maintaining a first sample at 80C and a second sample at 9~C for two hours, following which the samples were cooled and dried and their functional vitality compared with a regular vital gluten control.
The surprising result was that there was no signiflcant difference ~n vitality between the heat treated samples and the controls. It was from this surprising finding that the realiza-tion that wheat flour could be subjected to a relatively high temperature / short time process and, on that count alone, the gluten component would not be denatured.
Gluten Ball Test In order to determine rapidly and in a convenient manner, if an a-amylase sample is suitable to be used in the present process, a simple test was devised. A number of gluten balls were made by mixing commercial regular wheat gluten (lOg) with water (12g), the latter containing the enzyme to be evaluated for the desired efficacy. The balls were tested over a period of time by each being squeezed manually w~th the fingers . If the~enzyme under test had a sing'ficant protease activity, this manifested itself by causing the balls to soften and lose vitality. In the test, regular vital (80% protein) gluten scores 5 and O indicates no vitality as indicated by a lack of elasticity.Although this test is subjective to some extent, it is, in fact, quite reliable and able to be utilized by any person skilled in this art. Its reliability was confirmed in that, of the a-amylases tested, Calbiochem ~-amylase gave the best result - that enzyme did not affect the vitality of the gluten ball even following a five (5) hour incu~ation period at room temperature. Later analysisrevealed that that enzyme had a protease activity of only 0.4 AU/gm, by far the lowest of the enzymes tested and quite insufficient to adversely af~ect gluten vitality even over an extended period.
Another enzyme indicated by the test to be superior and therefore preferred was the Sigma ~-amylase.
~46~3~0 Process Parameter Evaluations Section A - using Termamyl 60L, supplied in liquid form having an activity of 60 K~u/~.
Temperature A series of experiments were conducted to determine the effect of temperature on the present process. The process was carried out at the following temperatures:
65C: 75C: 80C: 85C: 90C: 95C
The substrate was Glenrose flour ~Ogilvie ~ r Mills, Montreal) having the following composition:
Protein 9.53 Starch 82.30 Solubles 2.32 Moisture 11.88 On each occasion, 100 g of flour and 1 ml (equivalent to 0.05%) of enzyme were charged to the reaction cup. The time was noted for completion of the reaction to total starch disapp-earance, this being determlned by the standard iodine test for detecting starch. The result~ are given in Fig. 1.
The results show quite clearly that the time required for con-version reduces drastically as the temperature is increased especially to temperatures over 80C.
EnzYme Concentration Using the reactants as detailed above, a series of experiments were conducted to determine the amount of Termamyl 60L required for efficient conversion. The results are contained in the following Table 1 .
Table 1 _ Enzyme Concentration Temperature 0.5% ¦ 1.0~ ¦ 1.5 65C 120 min. 120 min.
80C 60 min. 36 min. 28 90C 11.5 min. 9.5 * Flour suspension not completely liquefied ~1~6~3VO
The results show that about 1% (by weight based on the flour) of the enzyme is effective. If the amount of enzyme is less,the tLme required at low temperatures becomes prohibitively long whilst at high temperatures the starch did not completely liquefy. Increasing the enzyme concentration gained little in terms of reduced conversion time. Moreover, the excess enzyme could actually be detrimental in that, if the enzyme used does have (even a small) protease activity, the total protease may be increased to a level where it may have significant adverse consequences on the gluten protein.
6uhstrate ~lour) Concen~r~tion A series of experiments were conducted to determine the effect of flour concentration on starch conversion efficiency using the Termamyl ~-amylase at a constant level of 1 ml enzyme solution/100 g ~uffaloflour; the concentration of the latter being varied as follows:
20~: 25%: 30~: 35%: and 40~
The results are included in the following Table ~
Flour conc. ~ 20 25 30 35 40 ._ ...
Iodine Reaction neg neg trace pos pos Syrup Weight (g) 264.0 253.3 241.0 209.4 188.3 op 17.27 21.69 35.80 29.20 32.13 Extract in Syrup (g) 45.6 54.9 62.2 61.1 60.5 . _ .
The re~ults show that at flour concentrations of about 35~ or more, starch liquifaction is inadequate, ~under the stated condittons which are within the optimum range for the enzyme ~n question), to the extent that separation of the gluten would prove impractical. The maximum of about 35~ of flour in the starting slurry ls generally applicable.
.The gluten products obtained in those experiments were analyzed and the results are given in Table 4 below:
l ml BAN enz~ e (b)2~ml BAN enzy~ë
40 min. at 80 C 22 mtn. at 80 C
... . .
Protein, % 55.19 67.38 Total lipid, % 8.83 11.27 Pentosan, % 23.96 9.68 Starch, % 15.18 4.73 Ash, ~ not determined 1.89 Both gluten products were ~unctionaland product (b) proved similar ln baking performance to xegular vital gluten.
Influence OfFlour TvPe on Products ~p1 B In this experiment Buffalo, Harvest Queen and Glenrose flours of the following compositions were processed according to the present in~ention:
. _ Component Buffalo Harvest Queen Glenrose . . . _ .__ Protein ~%) 14.14 12.20 9.53 Moisture (%) 9.5 11.37 11.88 Starch ~,db) 69.51 73.02 82.30 Solubles (%) 1.98 2.24 2.32 Ash (%)0.85 0.51 0.43 .
The BAN 120L ~-amylase was used in a concentration of 2mlflOOg flour and at 50C, and the resulting syrup and gluten separated by centrifugation in the usual manner.
Analysis details of the syrup and gluten products o~tained are contained in the following Ta~le 5 ~3 - 114t:~8~)0 Table 5 ~ufe~lo Queen Glenrose Syrup Degree 16.3 16.3 17.3 ~ Protein in syrup ~solids 3.2 3.1 2.5 Gluten Yield 17.2 14.3 10.7 .
Protein (d.b.) 67.7 68.2 61.2 Gluten Ball Test 1-2 0-1 Conclusions -(i) The Glenrose flour gave syrup having the highest plato value but the lowest gluten yield which was also the lowest in quality (vitality) of the three sample~.
(ii) The Buffalo flour gave the hlghest gluten yield.
(i~ and (li) are not unexpected since Glenrose is high in starch/low in protein whereas Buffalo i5 high in protein but relatively low in starch. However, the Buffalo-derived gluten also had the most vitality.
Section C
Experiments utilizing the Calbiochem enzyme supplied in crystalline form and having an activity of 1477 AU/mg.
(i) Enzyme Concentration A series of experiments utilizing increasing concentra-tions of enzyme were carried out, enzyme concentrations being:
1.146~)0 -0.025%: 0.050%: 0.075%: and 0.100%
The flour used was Buffalo and all the experiments were carried out at 70 C with a liquefaction time of sixty (6) minutes.
Results (i) At the 0.025% level, a positive iodine test indicated that the reaction was not completed.
(ii) The sample at the 0.~5% level gave a trace iodine test but all enzyme levels above 0.05% gave a negative results i.e. the reaction was complete.
(iii) Consideration of the syrup gravity values, yields of gluten fractions and protein contents of such fractions indicate that enzyme levels above 0.05% were adequate and, indeed, this was confirmed when the gluten ~all test indicated that the gluten obtained at the higher enzyme levels was in fact better, i.e.
more vital, etc.
~ii) pH Effect A ~eries of experiments were carried out utilizing the Calbiochem enzyme at the 0.05~ level in a Buffalo flour:water , (1:4) slurry at 70C for sixty (60) minutes liquefaction period, the pH of the slurries (adjusted using hydrochloric acid or sodium hydroxide as necessaryt increasing as follows: 4.5: 5.0:
5.5: 6.0: 6.5: and 7.
The results are included in Fig. ~ and show that the optimal pH for this enzyme is from 6 to 7 this giving the highest starch conversion tto a 16.5P syrup) and optimum gluten quality, both protein content t64-65~) and ~itality. Since flour/water su~pensionq ha~e a natural pH of about 6.05-6.10, no adjustment of pH is really requtred and throughout this specification all flour:water slurries have a pH of from 6.0 to 7.0 unless other-wise stated.
(iii) Full Process Again utilizing the Calbiochem enzyme, the process was carried out whereby 1200 g of Buffalo flour in a 1:4 aqueous slurry was acted upon by 0.6g of enzyme at about 70C for thirty (30) minutes.~ The process showing full mass balance is shown in Fig. 3 . As can be seen, some refinement to the basic process was made - in particular the initially obtained gluten fraction - 1~4~8~0 Gi was washed resulting in the generation of second gluten fraction G2, the wash water Wl being used as slurry make-up water, the G2 gluten fraction was comprised of two products as obtained from the centrifuge,whiCh products are subsequently freeze-dried as products Pt and Pb(~n summary,the products obtained are:
l Yield (g~ op ¦ % Protein (db) . _ , . .
Syrup Pl 4,896 16~72 Syrup P2 5~073 2.56 I (recirculated as I make-up) GlutenProduct, I 44.03 GlutenProductl ' 70.02 The major gluten product Product A had the following composition:
Co~ponent ~ (dry basis) Protein 70.2 Lipid 9.46 Starch 4-45 Ash 1.08 Pentosan 5.17 This product had a vitality, as measured by the gluten ball test, e~uivalent to regular vital gluten. Moreover, this was conf~rmed via bread baking trials - refer Section E below 1~
11468~)0 Effect of Washing With and Without Agitation of the Gluten Product It was noticed that some starch remained in the insolubilized gluten fraction. This fraction was, therefore, subjected to a washing step prior to separation by centrifugation.
The experiment used 50 m~ Calbiochem enzyme (with 5.5 mg CaCl2 and 2.75 mg NaCl) per 100 g flour, a conversion temperature of 70C and a period of thirty t30~ mlnutes.
The results are contained in Table 6.
Table 6 = Agitation ~aring Blender No Agitation _ t2 min/med tum~ .
Flour UsedBuffalo S.SP2 * B.uf.falo SSP2*
proYrUF Weight(g)409.0 405.0 407.2 404.0 op 16.74 16.32 16.63 16.30 ClarityHazy Cloudy Hazy Cloudy Gluten Weight(g) Products (nfnaltyPeri~ 4.25 4.00 2.55 3.35 effected) bottom13.20 14.60 17.5 18.0 (% db)77.12 78.22 68.76 68.56 Ball test4+ 4+ 4 4 The exper~ment was repeated on a larger scale and the results of the second experiment are contained in Table 7, the flow diagram with mass balance for the latter being shown in Fig. 4.
* ExFerimental hish extract blend of Ogilvie Flour Mills.
11~6~300 Table 7 Weight, g ¦ 412.3 Syrup Product degree plato,P ¦ 16.31 clarity ¦ clear . _ _ Gluten top layer Weight (g) 4.21 Product B
Moisture (%) 3.06 Protein (% db) 39.07 Total lipid 7.33 Pantosan ~ 25.17 Ash ' 1.95 Starch ~ 14.02 1.
Gluten bottom layer Weight (g) ~ 13.29 Product C
Moisture (%) 1.37 Protein (~ db) 75.96 Total lipid 10.16 Pentosan 2.33 Ash 1.02 Staxch 1.6 The results clearly show that the additional washing step or operation not only greatly improves the starch syrup/gluten separation efficiency but also gave a better gluten product.
Again, this was confirmed via baking trials using the products B and C of Table 7 - Section E.
(iv) Purification of Gluten Product Although the products, and in particular the gluten products, of the process of the present invention are intended to be utilized with little or no purification, the gluten products can be upgraded if desired.
~68~)0 For example, 100 g of Buffalo flour was treated with 400 g water containing 50 mg Calbiochem ~-amylase at 70C for thirty (30) minutes and the resulting syrup and gluten separated by centrifugation in the usual manner. The wet gluten obtained was dispersed in water (made up of 500 g) centrifuged; the clear wash water removed as was the top white (carbohydrate) layer.
That procedure was repeated five (5) times. The result was vital gluten having a protein content increased to 77.8%. Although the white l~yer was positlve to iodine and hence lncluded some starch, it was composed mainly of non-starch material.
Section D
Using Sigma 4-x crystallized enzyme having an activity of 1270 U/mg.
In a similar fashion to Section C(ii), a series of experiments were conducted using Sigma enzyme at the somg level, a Buffalo flour/water 1:4 slurry, liquefaction temperature of 70 C for a period of sixty t60) minutes. The pH values used were:
~468~)0 4.5: 5.0: 5.5: 6.0: 6.5: and 7.0 The results are givenin Fig. 5.
Once again the optimum range as far as starch conversion (about 14.75P syrup produced~ and better gluten, high protein content of 64-65% and good vitality, is concerned is for 6.0 to 7Ø
ComParison of Calbiochem and Siqma Enzymes In a series of experiments, these two enzymes gave very similar results, the only noticeable difference being that the Calbiochem had a somewhat higher activity.
section E
Evaluation of Gluten Products Three gluten products of the present invention, in particular those produced according to the procedure described in Section C ~iii) - Product A
Sectlon C ('iii) - Product B
Section C (iii) - Product C
were tested to determine their performance in breadmaking.
Three gluten products had the followinq chemical composition:
Table 8 .
Product "A" Product "B'' Product "C"
Washing with agitation No Yes Yes Dry gluten productBottom Layer Top LayerBottom Layer Protein content % 70.0 39.1 76.0 Total lipid % 9.5 7.3 10.1 Starch ~ 4.3 14.0 1.7 Ash 1.1 ¦ 2.0 1.0 Gluten Ball Test 4 0 1 4+
Yield of gluten % (d.b. 17.3 4.5 ~ 14.5 ~46800 Bread samples were produced using the recipe and procedure given below, regular vital gluten being the control.
Results (i) Gluten A could replace up to 60% of regular vital gluten in bread making without any noticeable change in either bread volume or internal structure, the protein level remaining the same. Greater than 60% replacement resulted in a decrease in loaf volume and a crumb having a coarse, immature appearance.
(ii) Procedure B could replace up to 25% of regular vital gluten without noticeable effect. At levels above 25%, the dough molding became light, poor "oven spring" was apparent and smaller loaf volumes were obtained.
It may be noted that Product B had a low protein content and scored very poorly in the gluten ball test and yet could still replace up to 25% of regular vital gluten, which is ver~
significant from a practical economic viewpoint.
(iii) Product C was, to all intent and purposes, the same as regular vital gluten obtained by conventional processes. It could replace up to 100% of regular vital gluten with no noticeable changes in loaf volume, etc.
As indicated above, a major aspect of the present inven-tion is the realization that a staple item of commerce, wheat flour, which is readily available at reasonable cost, can be rapidly converted into syrup and gluten products. Moreover, the proce5g i9 very flexible in that it can be tailored to produce syrup and gluten products for sepcific applications at relatively low cost. For example, carbohydrate syrups can, and are, used in the brewing industry as replacement for conventional adjuncts such as corn grits, etc. inter alia on account of convenience, consistency, etc. of the syrups. The syrups for use in this applications require a Plato ~alue of from about 10-16 P. More-over extreme purity, especially as regards clarity, is not of great importance. Such a product is ideally suited to being produced by the present process at low cost.
11468~0 -Turning to gluten products, an obvious application is in the baking industry where large amounts of gluten are used, both solely to improve the protein content of the flour and in some instances to improve the protein content and to assist the indigenous protein in fulfilling its traditional role. As stated, the gluten products produced according to the invention may range in functionality from poor to as good as regular vital gluten, depending mainly upon the choice of starting flour but also on whether the product, as obtained directly from the process, is subjected to a washing procedure. Even the less vital but still functional to ~ome extent gluten produced by the process of the present invention can be utilized to replace a significant propor-tion of the more expensive regular vital gluten as the baking trials demonstrate, and the present invention allows such pro-ducts to be produced relatively inexpensively and at will. As regards, the latter, the plant required to carry out the process of the invention, especially on a continuous basis, can be relatively inexpensive both in capital and to operate.
Moreover, it readily lends itself to bein operable on a "when required" basis. In other word~, there will be es~entially no delay between the demand for product syrup and/or gluten arising and the product being supplied. If the plant is operated to provide, as required, only one of the two products, then the other product can be collected and stored for shipment. It is anticipated that the plants would be custom built for a single location such as a single brewery or bakery and consequently no large inventory of starting flour or storage for product would be required. The manpower required for such a plant would be minimal and would be employed on other duties when the plant iQ not being operated.
Backqround of Invention Wheat flour comprises from about 70-85% starch, from about 9-16% proteinaceous material ~about 12 to 21% gluten), and a number of minor components such as hemicelluloses (pentosans) and ash. The gluten and starch, and starch derived products, especially syrups, are standard commercial items and are generally obtained from wheat flour by a variety of water washing procedures such as that described in Canadian Patent No. 1,037,474. The various processes are actually relatively inefficient solid:solid separations in that both gluten and starch are essentially insoluble in water and consequently, following formation of a wheat flour dough or batter, the solid starch is simply washed from the solid gluten which, as obtained under the processing conditions is in a rubbery/elastic condition in the form of lumps or the like. These current commercial proces3es require a large amount of water for batter preparation and an additional large quantity of water for the actual washing of the starch from the gluten. During the process, some of the soluble flour components, such as the pentosans, simple sugars, soluble proteins, as well as some of the starch, which is solubilized by indigenous enzymes (such as ~-amylase), dissolve in the water. However, such potentially u~eful compo-nents are then lost since they remain in the waste~water. This soluble fraction generally represents from 6 to 10% of the flour substance ahd hence is a significant loss.
Moreover, huge volumes of water are used in the process and consequently the waste waterS contain relatively small amounts (about 1~ ) of the soluble components. If the waste water is merely disposed of, i.e. run to sewer, it becomes a major source of water pollution and, in fact, public authorities are now frowning upon, if not banning, such practices. An alterna-tive is to dry, or at least increase the solids content of, this waste material, a very expensive proposition in view of the large energy requirements. A further complication is that many possible profitable uses for the solubles obtainable upon drying or concentrating are precluded because, in many instances, 114~i8~)0 the solubles are contaminated with a relatively large amount of processing aids, especially sodium chloride. ~s is well known, gluten is by far the most phys~cally functional non-animal protein and is highly valued on account of that characteristic.
However, that functionality or vitality is lost i.e. the gluten becomes denatured, in many ways and in particular by heat, and moreover starch gelatinizes in the presence of heat. Consequently, the varlous wheat flour washing processes are carried out at relatlvely low temperatures, generally about 30C, but in any event ~ignificantly below 60C which is widely considered to be the maximum to which gluten may be subjected during such pro-cessing if its desirable functional properties are to be retained.
Moreover, that temperature is also critical from the viewpoint of the starch in that the latter begins to gelatinize at about 55C. As a result, it would prove virtually impossible to separate a gelatinized starch component from the gluten component since the starch would gel thereby making it unsusceptible to being washed from the gluten.
As stated above, the gluten and starch have many tradi-tional uses, and historically many of these require that the pro-ducts be produced to exacting specifications. For example, gluten is generally produced as vital wheat gluten having a protein con-tent greater than 75% and usually in excess of 80% and in as functional a form as possible. As for starch in general, this is finding new markets as the starting material for the production of various syrups which are being used to an ever increasing extent in, for example, sugar replacement, baking and brewing areas. Purification of syrups is quite difficult and expensive but for many applications pure, for example clear and non-coloured, syrups are required. Consequently, since starting with a purer starch would produce a more pure syrup requiring a less extensive purification, commercially available starch as used to produce syrups is utilized in a pure state to produce the resulting commercially available syrups. In summary, the vast majority of commercially available gluten and starch syrups are produced to quite exacting specifications.
However, in many applications, such as brewing (where the present applicant is a large user of wheat starch and syrup derived therefrom),such exacting standards of quality are, in fact, not required butsuch products are used because, in many 6~0 instances, they are the only available suitable products. This results in increased costs to the user and, when the wider implications are considered, a waste of resources in the form of the energy, etc. required to purify the products, where purification is actually n~necessary~
Processes are known for enzymatically solubilizing and converting starch tovarious products. For example, United States Patent No. 2,583,451 teaches a process for producing dextrose comprising treating pure starch with ~-amylase under non-gelatinizing conditions at temperatures less than about 45C
and for periods in excess of forty-eight hours. United States Patent No. 3,922,201 teaches a similar process where granular starch is treated with a-amylase and glucose isomerase to produce levulose at a temperature below ~hich rapid gelatinization o~
the starch would take place. The examples teach process times of twenty-four hours. There are other known processes of a similar nature, but the process conditions and reactants presently utilized are such that if proteinaceous components, and in particular gluten,were present, then such would be significantly adver~ely affected; for example, in the case of wheat flour, gluten would be denatured and rendered useless, for example, for baking purposes; and indeed, might be solubilized to a lessor or greater extent and cause significant contamination of the syrup and derivatives thereof and render the products unusable.
An object of the present invention is to provide a process which, starting with readily available wheat flour, produce~ a wheat starch syrup and essentially vital wheat gluten rapidly and relatively inexpenslvely.
A further object is to provide such a process wherein substantially all of the wheat flour components are recovered and are incorporated into the process products.
Yet a further objective is to provide such a process in which waste waters, generally a major polluting factor, are totally eliminated while at the same time improving syrup yield.
68~)0 General Statement of Invention It has now been found that wheat gluten, either per se, or when present as the protein component of wheat flour, is not as susceptible to being adversely affected ~y heat as is presently widely believed in this art.
In more detail, it has been found that the starch fraction of wheat flour can be liquefied in the presence of the gluten com-ponent by treatment in aqueous suspension with certain a-amylase enzymes at significantly elevated temperatures in excess of about 65C for a period of not more than about sixty (60) minutes without functional denaturation of the gluten component occurring.
Thus, the original solid:solid system is thereby converted to a liquid:solid system which can readily be subjected to conven-tional separation techniques to provide the desired syrup and gluten products.
Detailed Statement of Invention According to the present invention there is provided a process for the production of ~yrup and vital gluten from wheat flour the process including the steps of:
forming a slurry comprising wheat flour, water and an ~-amylase enzyme which has substantially no protease activity, the slurry having a pH compatible wlth the activity of the specific enzyme employed tgenerallY 5 to 71 and contalning less than about 35%
by welght o~ wheat flour;
maintaining said slurry at a temperature of from 65C to about 95C with agitatlon for a period less than sixty (60)minutes but sufficient to liquefy 9ubstantially all of the starch component of the flour without signiflcantly adversely affecting the gluten component; and subsequently separating the solid gluten from the liquefied starch syrup.
The process proceeds as follows: the starch gelatinizes whereupon the ~-amylase commences to act thereon, breaking the starch polymer down into smaller chain units thus causing liquefac-tion which prevents the viscosity of the reaction mass from in-creasing excessively and the reaction mixture from gelling. The process is completed when the starch has been liquefied to such an extent that the gluten can readily be removed from the resulting syrup. In practice this is when the majority of 1 ~4~ )0 ~ 5 -the starch has been solubilized.
The rate of ltquefaction of the starch increases as the temperature increases. A major limiting factor is the properti-es of the specific ~-amylase enzyme chosen and in particular the temperature range, etc. at which lt operates most effecitvely and, of course, the temperature at which it would be heat-deactivated. Temperatures within the range of about 70C to about 95C and especlally between 80C to about 90C are preferred.
Although, the reaction mass does not significantly gel, the vlscosity does increase to an extent where the efficiency of the realitvely small amount of the enzyme in converting the starch may be significantly impaired. Agitation of the reaction mass is therefore effected. However, such agita-tion must be controlled since a high level thereof has been found to reduce the yield and quality, especially clarity, of the syrup.
To some extent, therefore, the degree/amount of agitation used depends also on the specific quality of syrup or gluten product required.
Allied to the above i9 the concentration of substrate flour in the reaction mass. It has been found that if the concen-tration of flour exceeds about 35% by weight, then the starch component is not sufficiently liquefled to be practically separated.
On the other hand, too dilute a slurry would increase the liqui-factlon time; reduce the capacity of the equipment or necessitate increasing the size of the equipment; increase handling and energy costs,etc. Also a major factor is that the present process lends itself to producing a solubilized starch product, which may be a ~yrup, and which incorporates substantially all the water (and solubles) and is useable, as ls, since the concentration of available carbohydrates is at commercially acceptable levels for many uses without evaporative at other concentration stages being necessary. Use of excessive amounts of water, i.e. very low flour concentrates, would result in the resulting syrup being of low carbohydrate concentration which would reduce its utility in some areas. Consequently, it is preferred that the starting flour slurry contain from about 15% to less than 30% flour. It should be em-phasized that the starting flour material is just that, namely wheat flour as obtained directly from the milling of wheat with no up-grading whatsoever being necessary. Consequently, it is readily ~146~
available and at reasonable cost. It is possible to process flour fractions upgraded in protein but these do not process as well and are obviously more expensive.
The pH of the reaction mass may vary within the quite wide limits and is essentially the pH at which the selected enzyme operates most effectively. However, it has been found that many suitable a-amylase enzymes operate optimally within the range of 6 - 7, and most wheat flour slurries~suspensions have a natural pH of between pH 6 to 7. Consequently, the reaction mass preferably has a pH of from 6 to 7.
The a-amylase enzyme used mNst be substantially free of protease activity and, obviously, have the ability to function at relatively elevated temperatures, in the presence of a ~tabilizing agent (such as calcium chloride and sodium chloride~
if necessary,albeit for only a relatively short period of time.
Moreover, a-amylases which are most active in the pH range of from 6 to 7 are preferred, sinc~ as stated, aqueous slurries of wheat flour have a natural p~ within that range and, consequently, no ad~ustment of the slurry pH is required.
Examples of such a-amylases include certain species of the Bacillus microorganisms such as 8acillus subtilis and Bacillus licheniformis. Specific examples of such enzymes are as follows: f~ 1~
æ (i~ Calbiochem a-amylase - supplied by Calbiochem Co San Diego, California;
(ii) Sigma a-amylase - a 4x crystallized enzyme supplied by S~gma Chemical Co., St. Louis, Missouri;
(iii) Ban1120L - suppl~ed by Novo Industries of Copenhagen, Denmark;
(iv) Tenase - available from Miles Laboratories Inc., Elkhart Indiana;
(v) HT-1000 - available from Miles Laboratories, Inc., Elkhart,~Indiana;
(vi) Termamyl 60L - also supplied by Novo Industries; and (vii) Takatherm1~ also available from Miles Laboratories Inc., Elkhart, Indiana.
ti) to (v) belng derived from ~. subtil~s and (vi) to ~vit being derived ~rom 3 licheniformis.
68~
The concentration of a-amylase enzyme required is relatively small and obviously depends on many factors, especially the activity thereof and the process temperature. As the specific experiments detailed later indicate, there appears to be a minimum concentration of enzyme if complete liquifaction is to be achieved and that minimum can easily be found via simple experiments as described herein. For example, the following concentrations have been found satisfactory.
(a) Ban 12~L - 1 ml to 2 ml /100 g flour, activity being 120 KN~g;
(b) Calbiochem - > 0.05~ by weight, activity being 1477 AU/g;
(c) Termamyl 60L - > O.5% by weight, activity being 60 KNU/g Of course, an amount of enzy;me of suitable activity must be used; in other words the activity and amount of enzyme must be suf~icient to effect the necessary solubiliation within the stated process parameters. That amount of enzyme is termed "the effective amountnand is readily determined for each sel~cted enzyme using the general lnformation given herein.
In the accompan~tng F~gures:
F~g. l is a curve of conversion t~me ~ersus temperature for Termamyl a-amylase;
Fig. 2 is an activity curve obtained using Calbiochem a-amylase and various pH values;
Fig. 3 is a flow sheet wlth mass balance of an embodiment of the invention using Calbiochem ~-amylase;
Fig. 4 is similar to Fig. 3 but wherein agitation of the gluten product is carried out; and Fig. 5 is a similar curve to Fig. 1 but wherein the enzyme is Sigma a-amylase.
:~468~0 The present invention will be further described withreference to, but not limited by, the following specific experiment:
Experimentation The process of the present invention as described in B the following Examples was carried out in a CHAIZ (Nancy, France) apparatus, orig~nally intended as a brewery laboratory mashing un~t. The apparatusincorporates a temperature controlled bath in whlch reactlon cups are lodged and allows for agitation of the reactants via a stirring mechanism which, during all the Examples, was set at 200 r.p.m.
Separation of the s~rup and gluten was effected by centrifugation using a SORVAL RC-3 HGA swinging buc~et head centrifuge. However, other conventional separation techniques such as filtration and even sedimentation can be used, the choice depending to some extent on the required degree of separation of the li~uid from the solid phase and the specific constitution of the liquefied starch and gluten product(s) required.
Procedure A predetermined quantity of flour (usuallylOOg) was mixed with water, (in the ratio of 1:4 unless otherwise stated) and the selected enzyme~ ~ where required in combination with an accompanying stabilizing mixture of calcium chloride and sodium chloride) was added. The resultlng suspension was plac~din the CHAIZ, where the temperature of the bath had been adjusted to the desired value and the stirring immediately started.
The mixture was held, with stirring, at the set temperature for the duration of the process, following which, any further reaction was arrested by rapid cooling. The mixture was then centrifuged to obtain the heavier gluten and as the top phase, the liquefied starch.
In an alternative embodiment, the gluten was re-washed and centrifuged again, the second liquid phase (comprising a very dilute liquefied starch being used as the flour slurry ma~e-up water.
The experimental w~rk carried out to delineate the effects of temperature on the process of the present invention showed that the process was ameniable to being effected over the range of from 70 to up to about 95C, the results at higher temperatures, whilst showing greater conversion efficiency in terms of ~46800 _ g reduced processing times, also indicated that the liquefied starch and gluten products were not signficantly altered from the products at the lower temperatures. Since the available equtpment was most conveniently operated at temperatures of about 70C to 80C with little effect on the experimental time required, evaluations of other process parameters such as enzyme concentration, etc.
were in many instances carried out at temperatures within that range.
In addition, standard operation of the CHAIZ unit allowed only for batch-type processes and consequently, much of the w~rk effected was of that type. However, all indications are that no problems of substance would be encountered on carrying out the process of the present invention on a continuous basis.
Indeed, from a commercial viewpoint, that would be the pr~ferred mode of operation and the design of suitable e~uipment would present little difficulty.
Process parameters which are important to the efficient carrying out of the proces~ of the present invention include:
(a) temperature ~b) enzyme concentration (c) flour (substrate) concentrate (d) pH
(e) flour type Each specific enzyme i9 unique to some extent as regards degree of activity, optimal processing conditions of pH, temperature, etc., and consequently, in practice the actual process, in terms of the ~pecific values of each parameter, will be tailored to suit the specific enzyme. The experimentation work detailed herein does not, obviously, provide such data for each and every possible ~-amylase. However, the extensive data is fully sufficient for a man skilled in the art to determine, with a few simple experiments, the processing conditions to be most advantageously used with any selected enzyme.
~6800 Effect o~ ~eat, per se, on ~ital ~heat ~-luten In this experiment the effect of heat, per se, on vital gluten was determined by reconstituting standard regular vital wheat gluten (obtained from IGP Limited, Montreal) with distilled water and, following reconstitution, maintaining a first sample at 80C and a second sample at 9~C for two hours, following which the samples were cooled and dried and their functional vitality compared with a regular vital gluten control.
The surprising result was that there was no signiflcant difference ~n vitality between the heat treated samples and the controls. It was from this surprising finding that the realiza-tion that wheat flour could be subjected to a relatively high temperature / short time process and, on that count alone, the gluten component would not be denatured.
Gluten Ball Test In order to determine rapidly and in a convenient manner, if an a-amylase sample is suitable to be used in the present process, a simple test was devised. A number of gluten balls were made by mixing commercial regular wheat gluten (lOg) with water (12g), the latter containing the enzyme to be evaluated for the desired efficacy. The balls were tested over a period of time by each being squeezed manually w~th the fingers . If the~enzyme under test had a sing'ficant protease activity, this manifested itself by causing the balls to soften and lose vitality. In the test, regular vital (80% protein) gluten scores 5 and O indicates no vitality as indicated by a lack of elasticity.Although this test is subjective to some extent, it is, in fact, quite reliable and able to be utilized by any person skilled in this art. Its reliability was confirmed in that, of the a-amylases tested, Calbiochem ~-amylase gave the best result - that enzyme did not affect the vitality of the gluten ball even following a five (5) hour incu~ation period at room temperature. Later analysisrevealed that that enzyme had a protease activity of only 0.4 AU/gm, by far the lowest of the enzymes tested and quite insufficient to adversely af~ect gluten vitality even over an extended period.
Another enzyme indicated by the test to be superior and therefore preferred was the Sigma ~-amylase.
~46~3~0 Process Parameter Evaluations Section A - using Termamyl 60L, supplied in liquid form having an activity of 60 K~u/~.
Temperature A series of experiments were conducted to determine the effect of temperature on the present process. The process was carried out at the following temperatures:
65C: 75C: 80C: 85C: 90C: 95C
The substrate was Glenrose flour ~Ogilvie ~ r Mills, Montreal) having the following composition:
Protein 9.53 Starch 82.30 Solubles 2.32 Moisture 11.88 On each occasion, 100 g of flour and 1 ml (equivalent to 0.05%) of enzyme were charged to the reaction cup. The time was noted for completion of the reaction to total starch disapp-earance, this being determlned by the standard iodine test for detecting starch. The result~ are given in Fig. 1.
The results show quite clearly that the time required for con-version reduces drastically as the temperature is increased especially to temperatures over 80C.
EnzYme Concentration Using the reactants as detailed above, a series of experiments were conducted to determine the amount of Termamyl 60L required for efficient conversion. The results are contained in the following Table 1 .
Table 1 _ Enzyme Concentration Temperature 0.5% ¦ 1.0~ ¦ 1.5 65C 120 min. 120 min.
80C 60 min. 36 min. 28 90C 11.5 min. 9.5 * Flour suspension not completely liquefied ~1~6~3VO
The results show that about 1% (by weight based on the flour) of the enzyme is effective. If the amount of enzyme is less,the tLme required at low temperatures becomes prohibitively long whilst at high temperatures the starch did not completely liquefy. Increasing the enzyme concentration gained little in terms of reduced conversion time. Moreover, the excess enzyme could actually be detrimental in that, if the enzyme used does have (even a small) protease activity, the total protease may be increased to a level where it may have significant adverse consequences on the gluten protein.
6uhstrate ~lour) Concen~r~tion A series of experiments were conducted to determine the effect of flour concentration on starch conversion efficiency using the Termamyl ~-amylase at a constant level of 1 ml enzyme solution/100 g ~uffaloflour; the concentration of the latter being varied as follows:
20~: 25%: 30~: 35%: and 40~
The results are included in the following Table ~
Flour conc. ~ 20 25 30 35 40 ._ ...
Iodine Reaction neg neg trace pos pos Syrup Weight (g) 264.0 253.3 241.0 209.4 188.3 op 17.27 21.69 35.80 29.20 32.13 Extract in Syrup (g) 45.6 54.9 62.2 61.1 60.5 . _ .
The re~ults show that at flour concentrations of about 35~ or more, starch liquifaction is inadequate, ~under the stated condittons which are within the optimum range for the enzyme ~n question), to the extent that separation of the gluten would prove impractical. The maximum of about 35~ of flour in the starting slurry ls generally applicable.
.The gluten products obtained in those experiments were analyzed and the results are given in Table 4 below:
l ml BAN enz~ e (b)2~ml BAN enzy~ë
40 min. at 80 C 22 mtn. at 80 C
... . .
Protein, % 55.19 67.38 Total lipid, % 8.83 11.27 Pentosan, % 23.96 9.68 Starch, % 15.18 4.73 Ash, ~ not determined 1.89 Both gluten products were ~unctionaland product (b) proved similar ln baking performance to xegular vital gluten.
Influence OfFlour TvPe on Products ~p1 B In this experiment Buffalo, Harvest Queen and Glenrose flours of the following compositions were processed according to the present in~ention:
. _ Component Buffalo Harvest Queen Glenrose . . . _ .__ Protein ~%) 14.14 12.20 9.53 Moisture (%) 9.5 11.37 11.88 Starch ~,db) 69.51 73.02 82.30 Solubles (%) 1.98 2.24 2.32 Ash (%)0.85 0.51 0.43 .
The BAN 120L ~-amylase was used in a concentration of 2mlflOOg flour and at 50C, and the resulting syrup and gluten separated by centrifugation in the usual manner.
Analysis details of the syrup and gluten products o~tained are contained in the following Ta~le 5 ~3 - 114t:~8~)0 Table 5 ~ufe~lo Queen Glenrose Syrup Degree 16.3 16.3 17.3 ~ Protein in syrup ~solids 3.2 3.1 2.5 Gluten Yield 17.2 14.3 10.7 .
Protein (d.b.) 67.7 68.2 61.2 Gluten Ball Test 1-2 0-1 Conclusions -(i) The Glenrose flour gave syrup having the highest plato value but the lowest gluten yield which was also the lowest in quality (vitality) of the three sample~.
(ii) The Buffalo flour gave the hlghest gluten yield.
(i~ and (li) are not unexpected since Glenrose is high in starch/low in protein whereas Buffalo i5 high in protein but relatively low in starch. However, the Buffalo-derived gluten also had the most vitality.
Section C
Experiments utilizing the Calbiochem enzyme supplied in crystalline form and having an activity of 1477 AU/mg.
(i) Enzyme Concentration A series of experiments utilizing increasing concentra-tions of enzyme were carried out, enzyme concentrations being:
1.146~)0 -0.025%: 0.050%: 0.075%: and 0.100%
The flour used was Buffalo and all the experiments were carried out at 70 C with a liquefaction time of sixty (6) minutes.
Results (i) At the 0.025% level, a positive iodine test indicated that the reaction was not completed.
(ii) The sample at the 0.~5% level gave a trace iodine test but all enzyme levels above 0.05% gave a negative results i.e. the reaction was complete.
(iii) Consideration of the syrup gravity values, yields of gluten fractions and protein contents of such fractions indicate that enzyme levels above 0.05% were adequate and, indeed, this was confirmed when the gluten ~all test indicated that the gluten obtained at the higher enzyme levels was in fact better, i.e.
more vital, etc.
~ii) pH Effect A ~eries of experiments were carried out utilizing the Calbiochem enzyme at the 0.05~ level in a Buffalo flour:water , (1:4) slurry at 70C for sixty (60) minutes liquefaction period, the pH of the slurries (adjusted using hydrochloric acid or sodium hydroxide as necessaryt increasing as follows: 4.5: 5.0:
5.5: 6.0: 6.5: and 7.
The results are included in Fig. ~ and show that the optimal pH for this enzyme is from 6 to 7 this giving the highest starch conversion tto a 16.5P syrup) and optimum gluten quality, both protein content t64-65~) and ~itality. Since flour/water su~pensionq ha~e a natural pH of about 6.05-6.10, no adjustment of pH is really requtred and throughout this specification all flour:water slurries have a pH of from 6.0 to 7.0 unless other-wise stated.
(iii) Full Process Again utilizing the Calbiochem enzyme, the process was carried out whereby 1200 g of Buffalo flour in a 1:4 aqueous slurry was acted upon by 0.6g of enzyme at about 70C for thirty (30) minutes.~ The process showing full mass balance is shown in Fig. 3 . As can be seen, some refinement to the basic process was made - in particular the initially obtained gluten fraction - 1~4~8~0 Gi was washed resulting in the generation of second gluten fraction G2, the wash water Wl being used as slurry make-up water, the G2 gluten fraction was comprised of two products as obtained from the centrifuge,whiCh products are subsequently freeze-dried as products Pt and Pb(~n summary,the products obtained are:
l Yield (g~ op ¦ % Protein (db) . _ , . .
Syrup Pl 4,896 16~72 Syrup P2 5~073 2.56 I (recirculated as I make-up) GlutenProduct, I 44.03 GlutenProductl ' 70.02 The major gluten product Product A had the following composition:
Co~ponent ~ (dry basis) Protein 70.2 Lipid 9.46 Starch 4-45 Ash 1.08 Pentosan 5.17 This product had a vitality, as measured by the gluten ball test, e~uivalent to regular vital gluten. Moreover, this was conf~rmed via bread baking trials - refer Section E below 1~
11468~)0 Effect of Washing With and Without Agitation of the Gluten Product It was noticed that some starch remained in the insolubilized gluten fraction. This fraction was, therefore, subjected to a washing step prior to separation by centrifugation.
The experiment used 50 m~ Calbiochem enzyme (with 5.5 mg CaCl2 and 2.75 mg NaCl) per 100 g flour, a conversion temperature of 70C and a period of thirty t30~ mlnutes.
The results are contained in Table 6.
Table 6 = Agitation ~aring Blender No Agitation _ t2 min/med tum~ .
Flour UsedBuffalo S.SP2 * B.uf.falo SSP2*
proYrUF Weight(g)409.0 405.0 407.2 404.0 op 16.74 16.32 16.63 16.30 ClarityHazy Cloudy Hazy Cloudy Gluten Weight(g) Products (nfnaltyPeri~ 4.25 4.00 2.55 3.35 effected) bottom13.20 14.60 17.5 18.0 (% db)77.12 78.22 68.76 68.56 Ball test4+ 4+ 4 4 The exper~ment was repeated on a larger scale and the results of the second experiment are contained in Table 7, the flow diagram with mass balance for the latter being shown in Fig. 4.
* ExFerimental hish extract blend of Ogilvie Flour Mills.
11~6~300 Table 7 Weight, g ¦ 412.3 Syrup Product degree plato,P ¦ 16.31 clarity ¦ clear . _ _ Gluten top layer Weight (g) 4.21 Product B
Moisture (%) 3.06 Protein (% db) 39.07 Total lipid 7.33 Pantosan ~ 25.17 Ash ' 1.95 Starch ~ 14.02 1.
Gluten bottom layer Weight (g) ~ 13.29 Product C
Moisture (%) 1.37 Protein (~ db) 75.96 Total lipid 10.16 Pentosan 2.33 Ash 1.02 Staxch 1.6 The results clearly show that the additional washing step or operation not only greatly improves the starch syrup/gluten separation efficiency but also gave a better gluten product.
Again, this was confirmed via baking trials using the products B and C of Table 7 - Section E.
(iv) Purification of Gluten Product Although the products, and in particular the gluten products, of the process of the present invention are intended to be utilized with little or no purification, the gluten products can be upgraded if desired.
~68~)0 For example, 100 g of Buffalo flour was treated with 400 g water containing 50 mg Calbiochem ~-amylase at 70C for thirty (30) minutes and the resulting syrup and gluten separated by centrifugation in the usual manner. The wet gluten obtained was dispersed in water (made up of 500 g) centrifuged; the clear wash water removed as was the top white (carbohydrate) layer.
That procedure was repeated five (5) times. The result was vital gluten having a protein content increased to 77.8%. Although the white l~yer was positlve to iodine and hence lncluded some starch, it was composed mainly of non-starch material.
Section D
Using Sigma 4-x crystallized enzyme having an activity of 1270 U/mg.
In a similar fashion to Section C(ii), a series of experiments were conducted using Sigma enzyme at the somg level, a Buffalo flour/water 1:4 slurry, liquefaction temperature of 70 C for a period of sixty t60) minutes. The pH values used were:
~468~)0 4.5: 5.0: 5.5: 6.0: 6.5: and 7.0 The results are givenin Fig. 5.
Once again the optimum range as far as starch conversion (about 14.75P syrup produced~ and better gluten, high protein content of 64-65% and good vitality, is concerned is for 6.0 to 7Ø
ComParison of Calbiochem and Siqma Enzymes In a series of experiments, these two enzymes gave very similar results, the only noticeable difference being that the Calbiochem had a somewhat higher activity.
section E
Evaluation of Gluten Products Three gluten products of the present invention, in particular those produced according to the procedure described in Section C ~iii) - Product A
Sectlon C ('iii) - Product B
Section C (iii) - Product C
were tested to determine their performance in breadmaking.
Three gluten products had the followinq chemical composition:
Table 8 .
Product "A" Product "B'' Product "C"
Washing with agitation No Yes Yes Dry gluten productBottom Layer Top LayerBottom Layer Protein content % 70.0 39.1 76.0 Total lipid % 9.5 7.3 10.1 Starch ~ 4.3 14.0 1.7 Ash 1.1 ¦ 2.0 1.0 Gluten Ball Test 4 0 1 4+
Yield of gluten % (d.b. 17.3 4.5 ~ 14.5 ~46800 Bread samples were produced using the recipe and procedure given below, regular vital gluten being the control.
Results (i) Gluten A could replace up to 60% of regular vital gluten in bread making without any noticeable change in either bread volume or internal structure, the protein level remaining the same. Greater than 60% replacement resulted in a decrease in loaf volume and a crumb having a coarse, immature appearance.
(ii) Procedure B could replace up to 25% of regular vital gluten without noticeable effect. At levels above 25%, the dough molding became light, poor "oven spring" was apparent and smaller loaf volumes were obtained.
It may be noted that Product B had a low protein content and scored very poorly in the gluten ball test and yet could still replace up to 25% of regular vital gluten, which is ver~
significant from a practical economic viewpoint.
(iii) Product C was, to all intent and purposes, the same as regular vital gluten obtained by conventional processes. It could replace up to 100% of regular vital gluten with no noticeable changes in loaf volume, etc.
As indicated above, a major aspect of the present inven-tion is the realization that a staple item of commerce, wheat flour, which is readily available at reasonable cost, can be rapidly converted into syrup and gluten products. Moreover, the proce5g i9 very flexible in that it can be tailored to produce syrup and gluten products for sepcific applications at relatively low cost. For example, carbohydrate syrups can, and are, used in the brewing industry as replacement for conventional adjuncts such as corn grits, etc. inter alia on account of convenience, consistency, etc. of the syrups. The syrups for use in this applications require a Plato ~alue of from about 10-16 P. More-over extreme purity, especially as regards clarity, is not of great importance. Such a product is ideally suited to being produced by the present process at low cost.
11468~0 -Turning to gluten products, an obvious application is in the baking industry where large amounts of gluten are used, both solely to improve the protein content of the flour and in some instances to improve the protein content and to assist the indigenous protein in fulfilling its traditional role. As stated, the gluten products produced according to the invention may range in functionality from poor to as good as regular vital gluten, depending mainly upon the choice of starting flour but also on whether the product, as obtained directly from the process, is subjected to a washing procedure. Even the less vital but still functional to ~ome extent gluten produced by the process of the present invention can be utilized to replace a significant propor-tion of the more expensive regular vital gluten as the baking trials demonstrate, and the present invention allows such pro-ducts to be produced relatively inexpensively and at will. As regards, the latter, the plant required to carry out the process of the invention, especially on a continuous basis, can be relatively inexpensive both in capital and to operate.
Moreover, it readily lends itself to bein operable on a "when required" basis. In other word~, there will be es~entially no delay between the demand for product syrup and/or gluten arising and the product being supplied. If the plant is operated to provide, as required, only one of the two products, then the other product can be collected and stored for shipment. It is anticipated that the plants would be custom built for a single location such as a single brewery or bakery and consequently no large inventory of starting flour or storage for product would be required. The manpower required for such a plant would be minimal and would be employed on other duties when the plant iQ not being operated.
Claims (22)
1. A process for the production of a solubilized starch product and functional wheat gluten from wheat flour comprising:
forming a slurry comprising wheat flour in an amount of less than 35% by weight of the slurry, water and an effective amount of .alpha.-amylase enzyme which exhibits substantially no protease activity, the wheat flour having a protein content of less than 20%
by weight, the slurry having a pH compatible with the activity of the specific enzyme employed, maintaining said slurry at a temperature of from about 65°C to about 100°C with agitation for a period less than sixty minutes but sufficient to liquefy substantially all of the starch components of the flour without adversely affecting the gluten component; and subsequently separating the solid gluten from the liquefied starch phase.
forming a slurry comprising wheat flour in an amount of less than 35% by weight of the slurry, water and an effective amount of .alpha.-amylase enzyme which exhibits substantially no protease activity, the wheat flour having a protein content of less than 20%
by weight, the slurry having a pH compatible with the activity of the specific enzyme employed, maintaining said slurry at a temperature of from about 65°C to about 100°C with agitation for a period less than sixty minutes but sufficient to liquefy substantially all of the starch components of the flour without adversely affecting the gluten component; and subsequently separating the solid gluten from the liquefied starch phase.
2. A process for the production of a solubilized starch product and functional wheat gluten from wheat flour comprising:
forming a slurry comprising wheat flour in an amount of from 20 to 30% by weight of the slurry: water, and an effective amount of an .alpha.-amylase enzyme which exhibits substantially no protease activity, the wheat flour having a protein content of less than 20% by weight, the slurry having a pH compatible with the activity of the specific enzyme employed;
maintaining said slurry at a temperature of from about 75°C to about 95°C with agitation for a period less than sixty minutes but sufficient to liquefy substantially all of the starch components of the flour without adversely affecting the gluten component; and subsequently separating the solid gluten from the liquefied starch phase.
forming a slurry comprising wheat flour in an amount of from 20 to 30% by weight of the slurry: water, and an effective amount of an .alpha.-amylase enzyme which exhibits substantially no protease activity, the wheat flour having a protein content of less than 20% by weight, the slurry having a pH compatible with the activity of the specific enzyme employed;
maintaining said slurry at a temperature of from about 75°C to about 95°C with agitation for a period less than sixty minutes but sufficient to liquefy substantially all of the starch components of the flour without adversely affecting the gluten component; and subsequently separating the solid gluten from the liquefied starch phase.
3. A process according to claim 1 or 2 wherein the temperature is between 80°C and 90°C.
4. A process according to claim 1 or 2 wherein the temperature is between 80°C and 100°C.
5. A process according to claim 1 or 2 wherein the pH of the slurry is from 6 to 7.
6. A process according to claim 1 or 2 wherein the enzyme treatment is continued for a period of time sufficient to convert the starch component into a starch syrup.
7. A process according to claim 1 or 2 wherein the gluten is removed from the solubilized starch phase by centrifugation.
8. A process according to claim 1 or 2 wherein said slurry is maintained at the elevated temperature for less than thirty minutes.
9. A process according to claim 1 or 2 wherein said slurry is maintained at the elevated temperature for less than fifteen minutes.
10. A process according to claim 1 wherein the slurry comprises 20 to 30% by weight of flour; the pH is 6 to 7: and the slurry is maintained at a temperature of between 80°C to about 100°C for less than thirty minutes.
11. A process according to claim 1, 2 or 10 wherein said slurry is maintained at the elevated temperature for less than 5 minutes.
12. A process according to claim 1, 2 or 10 wherein said slurry is maintained at the elevated temperature for from 1 to 5 minutes.
13. A process according to claim 1, 2 or 10 wherein the gluten product is purified by contact with water.
14. A process according to claim 1, 2 or 10 wherein the .alpha.-amylase is derived from B. Lichemiformis.
15. A process for the production of a starch syrup and functional gluten from wheat starch comprising forming a slurry comprising wheat flour, water and an .alpha.-amylase which exhibits substantially no protease activity, the wheat flour having a protein content of less than 20% by weight, the slurry having a pH
compatible with the activity of the specific enzyme employed and containing from 15 to 30% by weight of wheat flour;
maintaining said slurry at a temperature of between 80°C
and 100°C with agitation for a period less than sixty minutes but sufficient to liquefy substantially all of the starch components of flour to form a starch syrup without significantly adversely affecting the gluten component; and subsequently separating the solid gluten from the liquefied starch phase.
compatible with the activity of the specific enzyme employed and containing from 15 to 30% by weight of wheat flour;
maintaining said slurry at a temperature of between 80°C
and 100°C with agitation for a period less than sixty minutes but sufficient to liquefy substantially all of the starch components of flour to form a starch syrup without significantly adversely affecting the gluten component; and subsequently separating the solid gluten from the liquefied starch phase.
16. A process according to claim 15 wherein said slurry is maintained at a temperature of between 80°C and 95°C.
17. A process according to claim 15 wherein said slurry is maintained at a temperature of between 80°C and 80°C.
18. A process according to claim 15 wherein said slurry is maintained at the elevated temperature for a period less than thirty minutes.
18. A process according to claim 15 wherein said slurry is maintained at the elevated temperature for a period less than thirty minutes.
18. A process according to claim 16 wherein said slurry is maintained at the elevated temperature for a period less than about fifteen minutes.
19. A process according to claim 12 wherein said slurry is maintained at the elevated temperature for a period less than about five minutes.
20. A process according to claim 1, 2 or 10 wherein the gluten product is purified by contact with water.
21. A process according to claim 15 wherein the .alpha.-amylase is derived from B. Licheniformis.
22. A brewing process which utilizes a carbohydrate adjunct, the improvement comprising providing as the adjunct a solubilized starch produced by the process according to claim 1, 2 or 15.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000345671A CA1146800A (en) | 1980-02-13 | 1980-02-13 | Enzymatic treatment of wheat flour |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000345671A CA1146800A (en) | 1980-02-13 | 1980-02-13 | Enzymatic treatment of wheat flour |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1146800A true CA1146800A (en) | 1983-05-24 |
Family
ID=4116244
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000345671A Expired CA1146800A (en) | 1980-02-13 | 1980-02-13 | Enzymatic treatment of wheat flour |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1146800A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2681219A1 (en) * | 1991-09-16 | 1993-03-19 | Ard Sa | LIPIDOPROTEIC PRODUCT DERIVED FROM WHEAT FLOUR, PREPARATION METHOD AND APPLICATIONS THEREOF. |
| CN113907255A (en) * | 2021-10-25 | 2022-01-11 | 广东以琳食品有限公司 | Medium-temperature enzymolysis process and device for preparing flour special for shredded cake |
-
1980
- 1980-02-13 CA CA000345671A patent/CA1146800A/en not_active Expired
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2681219A1 (en) * | 1991-09-16 | 1993-03-19 | Ard Sa | LIPIDOPROTEIC PRODUCT DERIVED FROM WHEAT FLOUR, PREPARATION METHOD AND APPLICATIONS THEREOF. |
| EP0533512A1 (en) * | 1991-09-16 | 1993-03-24 | Agro Industrie Recherches Et Developpements (A.R.D.) | Lipoprotein containing product derived from wheat flour, process to prepare it, and applications thereof |
| CN113907255A (en) * | 2021-10-25 | 2022-01-11 | 广东以琳食品有限公司 | Medium-temperature enzymolysis process and device for preparing flour special for shredded cake |
| CN113907255B (en) * | 2021-10-25 | 2024-04-19 | 广东以琳食品有限公司 | Medium-temperature enzymolysis process and device for preparing flour special for hand-held cakes |
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