(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(19) World Intellectual Property

Organization
International Bureau
(10) International Publication Number
(43) International Publication Date WO 2015/131226 Al
11 September 2015 (11.09.2015) P O PCT

(51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every
A23J3/34 (2006.01) A23L 1/305 (2006.01) kind of national protection available): AE, AG, AL, AM,
A23J3/18 (2006.01) A23L 2/66 (2006.01) AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY,
C12P 21/06 (2006.01) A21D 2/26 (2006.01) BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM,
DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT,
(21) International Application Number:
HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR,
PCT/AU201 5/0001 13 KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG,
(22) International Filing Date: MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM,
2 March 2015 (02.03.2015) PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC,
SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN,
(25) Filing Language: English TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
(26) Publication Language: English
(84) Designated States (unless otherwise indicated, for every
(30) Priority Data: kind of regional protection available): ARIPO (BW, GH,
2014900728 5 March 2014 (05.03.2014) AU GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ,
TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU,
(71) Applicant (for all designated States except US): SHOAL- TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE,
HAVEN STARCHES PTY LTD [AU/AU]; 36 Bolong DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU,
Road, Bomaderry, NSW 2541 (AU). LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK,
SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ,
(72) Inventors; and
GW, KM, ML, MR, NE, SN, TD, TG).
(71) Applicants (for US only): PEARCE, Robert, John
[AU/AU]; 1 James Lane, Kiama, NSW 2533 (AU). BAR- Published:
RIE, Amy, Lee [AU/AU]; 5 Hanover Close, South Nowra,
— with international search report (Art. 21(3))
NSW 2541 (AU).
(74) Agent: ANDERSON-TAYLOR, Michael; P.O. Box 710,
Cronulla, NSW 2230 (AU).

-
© (54) Title: FUNCTIONAL INGREDIENTS FROM ENZYME-MODIFIED WHEAT PROTEINS
(57) Abstract: A method of producing an ingredient material for use in food, said method including the steps of transforming a par-
ent gluten complex by two or more enzymatic processes whereby said ingredient material displays different physical and functional
properties to said parent gluten complex.
 FUNCTIONAL INGREDIENTS FROM ENZYME-MODIFIED
WHEAT PROTEINS

FIELD OF THE INVENTION

The present invention relates to novel products derived from wheat gluten that can be
used advantageously as food ingredients in a range of foods additional to traditional
uses for gluten as in bakery goods, pasta and noodles. Additionally the present
invention relates to the process of making the aforesaid novel products and
applications for such novel products in foods.

BACKGROUND TO THE INVENTION

Wheat protein occurs mostly as a complex of gliadin and glutenin proteins which
when extracted from wheat flour with water forms a water-insoluble, viscoelastic
mass known as gluten. It is this property and material that renders wheat flour
uniquely suitable for making leavened bread, pasta, noodles and other food items
prepared from a wheat flour-based dough or batter. It is also this material and property
that restricts the use of wheat protein in most other manufactured food formulations.

As the protein most-extensively-grown globally for human food and with the globe
facing a food and protein shortage, there is a need to discover and develop wheat
protein derivatives to find wider usage in more specialised and versatile ingredient
forms. To this end for more than 20 years researchers have sought physical, chemical
and biological solutions to this challenge.

Thermal treatment of gluten, often traded as Vital Wheat Gluten, brings about the
well-established change to its structure and properties known as devitalisation. In this
process, elasticity and cohesiveness is lost and the protein mass shows a higher
affinity for water due to changes in the secondary, tertiary and quaternary structures
of the protein complex. Apart from use as a proteinaceous filler in some foods
especially meat sausage-type products, as an ingredient devitalised gluten offers little
advantageous functionality.

While the structure of the gluten complex is not fully understood, several models have
been proposed and in every one, disulphide cross links play an important role in
maintaining structural integrity and in conferring aspects of strength and elasticity to
the viscoelastic behaviour. Consequently, chemical treatments of gluten to break or
realign disulphide cross links have been described to change and enhance the
properties of gluten, see for example, Shewry, P.R. & Mifiin, B.J. (1985) Seed storage
proteins of economically important cereals Adv.Cer.Sci Technol 7,1-83

Wheat gluten is unique amongst proteins in its viscoelastic properties that are related
to the primary structures of the separate proteins of the gluten complex. The
uniqueness in composition is conferred via the extremely high content of the amino
acid glutamine representing more than one-third of all amino acids. Glutamine side
chains off the main peptide backbone of the proteins confer a high degree of
hydrophilicity but a low level of electrically charged sites on the protein surface.
Together wit h an high content of the structure-imposing amino acid, proline, and a
quite high level of apolar amino acid side chains and low content of ionisable amino
acid side chains results in high water affinity but very low water solubility.

Understandably, glutamine side chains have been the target for modification of wheat
gluten for enhancement of functional properties. Deamidation of glutamine results in
the formation of glutamic acid, an ionisable amino acid side chain which is also a
natural but non-essential amino acid, so the intrinsic nutritional value of the protein is
not changed. The process of chemical deamidation via acid- or alkali- catalysed
hydrolysis has been reviewed by William E. Riha III, Henry V Izzo, Jie Zhang and
Chi-Tang Ho (1996) Nonenzymatic Deamidation of Food Proteins Critical Reviews in
Food Science and Nutrition, 36(3), 225-255.

Deamidation of wheat proteins has been described by Matsudomi,N., Kato, A and

Kobayashi, K . (19820 Conformation and properties of deamidated gluten.
Agricultural and Biological Chemistry 46(6), 1583-1586.; Mimouni, B., Raymond,
J., Merledesnoyers, A.M. Azanza, J.L and Ducastaing, A.(1994) Combined acid
deamidation and enzymatic hydrolysis for improvement of the functional properties of
wheat gluten. Journal of Cereal Science 20(2) 153-1 65); Ahmedna, M.,
Prinyawiwatkul, W. and Rao, R.M. (1999) Solubilised wheat protein isolate;
functional properties and potential food applications. Journal of Agricultural and Food
Chemistry, 47(4), 1340-1345, and shown to markedly improve its solubility.
Deamidated wheat protein has been shown also to have good emulsification properties
comparable to other food proteins used as emulsifiers including casein, casemates and
soy protein isolate by Ahmedna, M., Prinyawiwatkul, W. and Rao, R.M. (1999)
Solubilised wheat protein isolate; functional properties and potential food
applications. Journal of Agricultural and Food Chemistry, 47(4), 1340-1345, and by
Webb, M.R. , Naeem, H.A. and Schmidt, K.A. (2002) Food protein functionality in a
liquid system; a comparison of deamidated wheat gluten with dairy and soy proteins
Journal of Food Science 67(8) 2896-2902, and the mechanism of emulsion
stabilisation by deamidated wheat protein has been described by Li Day, Mi Xu, Leif
Lundin and Tim j . Wooster (2009) Interfacial properties of deamidated wheat protein
in relation to its ability to stabilise oil-in-water emulsions. Food Hydrocolloids April,
1-10

Chemically-deamidated wheat proteins have been available in the global marketplace

for a number of years; early popularity due to excellent functionality properties,
particularly improved solubility, as food ingredients gave way to other materials as
chemically deamidated wheat protein became less well regarded due to unpleasant
flavour generation and to acid-deamidated wheat protein products being implicated in
new allergen sensitivities.

As the advantageous improved solubility was counteracted by adverse flavour and

health issues, alternative means of modifying gluten have been investigated and
disclosed. US Patent 6,610,334 discloses the use of thiol redox proteins for reducing
protein intramolecular disulphide bonds for improving the quality of cereal products,
dough and baked goods.

Enzyme treatment of proteins with protease enzymes has for some time been used to
increase their solubility as described by J . Adler-Nissen (1976) Enzymic hydrolysis of
proteins for increased solubility. Journal of Agricultural and Food Chemistry 24(6)
1090-1093. More recently, Qi Wei and He Zhimin (2006) Enzymatic hydrolysis of
proteins: mechanism and kinetic model. Chem. China 3, 308-3 14, have described a
mechanism and kinetic model for the enzymatic hydrolysis of proteins particularly in
regard to the release of active peptides from proteins.
Xiang Dong Sun (201 1) describes the enzymatic hydrolysis of soy proteins in
Enzymatic Hydrolysis of Soy Protein and the Hydrolysates Utilisation International
Journal of Food Science & Technology 46(2),2447-2459 and the advantageous
utilisation of soy protein hydrolysates in changing functional properties and
improving nutrition. US Patent Xiang Dong Sun (201 1) describes the enzymatic
hydrolysis of soy proteins in Enzymatic Hydrolysis of Soy Protein and the
Hydrolysates Utilisation International Journal of Food Science & Technology
46(2),2447-2459 discloses a partial hydrolysate of whey protein which contains active
peptides but does not have a bitter flavour. US Patent 8,101,377 discloses an
alternative method of controlling flavour and minimising bitterness in dairy protein
hydrolysates by commencing the process with protein in basic solution at pH about
10.4, cooling the solution and then adding protease enzyme that is allowed to function
over an extended period.

In US Patent 5,945,299 is disclosed a process for producing wheat protein

hydrolysates in a multi-stage hydrolysis with both a proteinase and a peptidase in
order to prevent instability in solution recognised as clouding. By this process a more
favourable molecular weight distribution of peptides is achieved. The process includes
use of a proteinase first at acidic pH then at alkaline pH and thirdly with peptidases at
pH6-7.

US Patent 6,036,983 discloses a method of obtaining protein hydrolysates useful as

flavouring agents. It is well known that extensive hydrolysis of proteinaceous
materials results in flavoursome products that may be used in soups, sauces and
seasonings. Furthermore, it is known that the amino acid glutamine (gin) is almost
tasteless whereas glutamic acid (glu) whether free or peptide bound plays an
important role for the flavour and palatability of protein hydrolysates. The process of
deamidation converts glutamine to glutamic acid and can be achieved by non-
enzymatic hydrolysis with acids or alkalis as described above. US Patent 6,036,983
teaches the use of of peptidoglutaminases such as Peptidyl-glutaminase EC 3.5.1.43
or Protein-glutamine glutaminase EC3.5.1.44 for enzymatic deamidation of peptides.

US Patent No 3,857,967 discloses a process for the preparation of a food or beverage

with a peptidoglutaminase from Bacillus circiilans but in order to achieve the greatest
degree of deamidation , US Patent No 3,857,967 teaches initial degradation of the
proteinaceous substrate by use of non-specific endo- and/ exo-peptidases

Mimouni et al Mimouni, B., Raymond, J., Merledesnoyers, A.M. Azanza, J.L and
Ducastaing, A.(1994) Combined acid deamidation and enzymatic hydrolysis for
improvement of the functional properties of wheat gluten. Journal of Cereal Science
20(2)153-165, describe a combined acid deamidation and enzymatic proteolysis using
non-specific endo-peptidases.

US Patent No 7,846,709 discloses a protein-deamidating enzyme recovered from

Chryseobacterium proteolyticum, the gene encoding for the same and a process for its
production. The enzyme is described further in Yamaguchi, S . & Yokoe, M . (2000) A
novel protein deamidating enzyme from Chryseobacterium proteolyticum sp. , a
newly isolated bacterium from soil. Appl. Environ. Microbiol. 66, 3337-3343.

The action of protein-glutaminase has been studied on several proteins: Gu,

Y.S.,Matsumura, Y., Yamaguchi, S . & Mori, T. (2001) Action of protein-glutaminase
on alpha-lactalbumin in the native and molten globule states J. Agric. Food Chem.
49, 5999-6005; Yong,Y.H., Yamaguch, S., Gu, Y.S.,Mori, T, & Matsumura, Y.
(2004) Effects of enzymatic deamidation by protein-glutaminase on structure and
functional properties of alpha-zein. Yong, Y.H., Yamaguchi, S . & Matsamura, Y .
(2006) Effects of enzymatic deamidation by protein-glutaminase on structure and
functional properties of wheat gluten. J . Agric. Food Chem. 54, 6034-6040 showed
that water-insoluble gluten was able to be deamidated up to 72% in 30 hours and
resulted in increase in solubility and improvement of emulsification properties. It was
suggested that allergenicity of deamidated gluten was decreased markedly as
deamidation extent was increased.

US Patent No 7,008,653 discloses a method for deamidat ion of milk protein and a
method for denaturation of milk protein by using a deamidating enzyme wherein the
enzyme is that described in US Patent No 7,846,709. US Patent No 7,008,709 also
discloses that said enzyme has a deamidating effect on a protein having a molecular
weight of 10,000 or more. Casein proteins were more readily deamidated than whey
proteins.
US Patent No 7,947,315 discloses a method for providing a dairy product smooth oral
sensation and suppressed acidic taste and bitter taste and a method for manufacturing
the same wherein a protein deamidating enzyme is added to raw milk to act on the
milk protein in the raw milk.

US Patent Application No 20130236627 discloses coffee whiteners, prepared by using

a casein-containing milk protein solution that has been deamidated with a protein
deamidating enzyme, exhibiting excellent storage stability and dispersibility in coffee
without the use of synthetic emulsifiers

OBJECT OF THE INVENTION

It is an object of the present invention to provide products obtained by novel

transformations of the gluten complex into ingredient materials having minimum
flavour and bland colour properties that display different physical and functional
properties from the parent gluten complex which enable said transformed materials to
be utilised advantageously as functional ingredients in a wide variety of minimally
flavoured food applications.

SUMMARY OF THE INVENTION

Accordingly said invention discloses A method of producing an ingredient material

for use in a food product, said method including the steps of transforming a parent
gluten complex by two or more enzymatic processes whereby said ingredient material
displays different physical and functional properties to said parent gluten complex.

In another aspect said invention further discloses a food product which includes an
ingredient material produced by the above method.

Preferably additional physical or chemical treatments may be included to further

modify the functional properties of the product.

In one of the processes the gluten complex is preferably fragmented by enzymatic

hydrolysis to an extent determined by the application for the product; in the second
process the interactive surface of the gluten fragments is preferably changed by
enzymatic hydrolysis of a proportion of the glutamine constituent of the peptides and
converting it to glutamic acid to an extent determined by the application for the
product.

In a further preferred form of the invention products are disclosed resulting from
additional transformation of the said transformed gluten fragments by additional
physical and chemical processes that may further modify said transformed gluten
fragments for advantageous outcomes.

In a furt her preferred form of the invention products are disclosed resulting from
additional transformation of the said transformed gluten fragments by additional
physical and chemical processes that may be applied between sequential application
of the two enzymatic processes that may further modify said transformed gluten
fragments for advantageous outcomes. More specifically, gluten fragments otherwise
referred to as gluten-derived peptides or gluten peptides may be separated according
to size or charge or may be separated according to solubility or may be separated
according to another physical or chemical property of the gluten peptides

In a further preferred form of the invention products are disclosed resulting from
modification of the parent gluten complex by physical and/or chemical processes prior
to transformation of the said modified gluten complex by means of the aforesaid
enzymatic processes.

In a further preferred form of the invention a process is disclosed that includes the
transformation of gluten by at least two enzymatic processes occurring concurrently
or sequentially. In one of the processes the gluten complex is fragmented by
enzymatic hydrolysis to an extent determined by the application for the product ; in
the second process the interactive surface of the gluten fragments is changed by
enzymatic hydrolysis of a proportion of the glutamine constituent of the fragments
and converting it to glutamic acid to an extent determined by the application for the
product.

In a further preferred form of the invention a process is disclosed that additional

transformation of the said transformed gluten fragments by additional physical and
chemical processes that may further modify said transformed gluten fragments for
advantageous outcomes according to the application for the product.

In a further preferred fonn of the invention products are disclosed resulting from
additional preparative processes applied to the said transformed gluten fragments by
physical means following the two enzymatic processes that may realise advantageous
outcomes. More specifically, transformed gluten fragments otherwise referred to as
transformed gluten-derived peptides or transformed gluten peptides may be separated
according to size or charge or may be separated according to solubility or may be
separated according to another physical or chemical property of the transformed
gluten peptides and may be dried by any suitable means.

In a further preferred fonn of the invention it is disclosed that products constituted by

and containing said novel transformed gluten fragments are utilised as ingredients in
food systems providing unique and novel physical and chemical properties. Said food
systems may include but not limited to:
novel foods arising from a dough or batter in which the elasticity, extensibility
viscosity or other functional property importantly described for such food systems is
enhanced;
novel manufactured meat products or meat analogue products in which the inclusion
of water and fat components is enhanced
novel liquid foods and beverages in which the inclusion of water and fat components
is enhanced
novel manufactured non-dairy fat powder and non-dairy creamer food products

Non-limiting examples of the invention that identify novel products, novel processes
and applications for said novel products are as follows:-

Example 1 Enzymatic proteolysis of vital wheat gluten followed by separation of

soluble from insoluble peptides followed by enzymatic deamidation of soluble
peptides

A commercial tryptic-like enzyme of fungal origin was dispersed in warm water

(60°C) in an amount being by weight 0.5% of the weight of dried vital wheat gluten to
be processed. The dry vital wheat gluten was added with vigorous stirring such that
the final content of gluten in the dispersion was 30% with the pH maintained between
5.8 and 6.2. After 1.5 hours when the amount of protein as enzyme-hydrolysed
peptides had reached 70%, the suspension was heated to 90°C and held at that
temperature for at least 1 minute to deactivate the proteolytic enzyme and prevent
any further fragmentation or development of bitter flavour. After cooling to 60°C the
suspension was centrifuged at 2000 x g for 3 minutes and the almost clear supernatant
separated from the insoluble sedimented material.

The solubilised peptides in the supernatant were then treated with protein-deamidase
enzyme (PG 50, Amano Enzymes) at 50°C with enzyme being dosed at 0.5% by
weight of solubilised gluten peptides maintained at pH between 5.8 and 6.2 for 5
hours. The solution was heated to 80°C to inactivate the deamidase enzyme and
cooled to 50°C, Dried product was obtained by spray drying the treated solution
directly.

Dried solubilised and deamidated product was analysed for protein content, moisture,
and ash content using standard methods of analysis. Extent of deamidation was
estimated from release of ammonia and confirmed by amino acid analysis after
complete enzymatic hydrolysis of the peptides.

Concentration of peptides in supernatant = 22%

Extent of solubilisation of gluten = 73%

Analysis by size-exclusion HPLC of fragmentation of gluten and molecular weight

distribution of soluble wheat peptides arising .

A 1% w/w solution of solubilised wheat peptides in the supernatant as aforesaid was

applied to a size exclusion HPLC column system constituted by 2 x TSK-3000
columns in series and eluted with water. Eluting peptides were detected and quantified
using a UV-detector system. The HPLC column system was calibrated with standard
reference proteins of known molecular weight and used to estimate molecular weights
eluting as a function of elution volume. Figure 1 shows an HPLC chromatogram
obtained by this procedure and in Table 1 is shown the estimation of peptide
molecular weights represented by the peaks shown on the HPLC chromatogram
(Figure 1) calculated relative to a calibration curve constructed using data from the
elution of standard proteins of known molecular weight. Computation of area under
each peak provides an estimation of the quantity of each peak. Relative quantities of
peptides in sequential size distribution bands are shown in Table 2 .

Table 1 Estimation of molecular weight distribution of soluble wheat peptides by

HPLC analysis

Ammonia release and extent of deamidation determined by Kjeldahl method

Progress of deamidation by protein deamidase was determined by sampling the

aforesaid reaction mixture at intervals, stopping further deamidation by heat
inactivating the enzyme as aforesaid and measuring the quantity of free ammonia
arisen from the deamidation reaction by a standard Kjeldahl method. Table 3 shows
the extent of ammonia released during the progress of the reaction.
Total potential deamidation was estimated by acid hydrolysis of the sample in 0 . M
sulphuric acid at 95°C for 30 minutes followed by measurement of the amount of
ammonia released. Comparison of amount of enzyme-released ammonia to total
potential provided an estimate of extent of deamidation as shown also in Table 3 .

Table 3 Estimation of extent of deamidation of soluble wheat peptides

Extent of deamidation determined by amino acid analysis

Confirmation of the extent of deamidation was achieved by amino acid analysis of the
untreated peptide preparation and the protein deamidase-treated preparation and
comparison of the sum of glutamic acid and aspartic acid content with the sum of
glutamine and asparagine contents in each preparation. Results of amino acid analyses
are shown in Table 4
Table 4 . Amino acid analysis of deamidated soluble wheat peptides and
deamidated soluble wheat peptides after total enzymatic hydrolysis

Example 2 . Enzymatic proteolysis of vital wheat gluten followed by separation of

soluble from insoluble peptides followed by enzymatic deamidation of insoluble
peptides

A commercial tryptic-like enzyme of fungal origin was dispersed in warm water

(60°C) in an amount being by weight 0.5% of the weight of dried vital wheat gluten to
be processed. The dry vital wheat gluten was added with vigorous stirring such that
the final content of gluten in the dispersion was 30% with the pH maintained between
5.8 and 6.2. After 1.5 hours when the amount of protein as enzyme-hydrolysed
peptides had reached 70%, the suspension was heated to 90°C and held at that
temperature for at least 1 minute to deactivate the proteolytic enzyme and prevent any
further fragmentation or development of bitter flavour. After cooling to 60°C the
suspension was centrifuged at 2000 x g for 3 minutes and the almost clear supernatant
separated from the insoluble sedimented material.

The insoluble peptides in the sediment were dispersed as a 15% w/w suspension then
treated with protein-deamidase enzyme (PG 50, Amano Enzymes) at 50°C with
enzyme being dosed at 1.0% by weight of solubilised gluten peptides maintained at
pH between 5.8 and 6.2 for 8 hours. The solution was heated to 80°C to inactivate the
deamidase enzyme and cooled to 50°C. Dried product was obtained by spray drying
the treated solution directly.

Dried insoluble peptide product and the corresponding deamidated product were
analysed for protein content, moisture and ash content using standard methods of
analysis. Extent of deamidation was estimated from release of ammonia and
confirmed by amino acid analysis after complete enzymatic hydrolysis of the
peptides.

Extent of solubilisation of gluten = 73%

Proportion of peptides as insoluble sediment = 27%

Ammonia release and extent of deamidation determined by Kjeldahl method

Progress of deamidation by protein deamidase was determined by sampling the

aforesaid reaction mixture at intervals, stopping further deamidation by heat
inactivating the enzyme as aforesaid and measuring the quantity of free ammonia
arisen from the deamidation reaction by a standard Kjeldahl method as in Example 1.

The total amount of amide-nitrogen in the original insoluble peptide sample was
estimated by acid hydrolysis of the sample in 0 . 1M sulphuric acid at 95°C for 30
minutes followed by measurement of the amount of ammonia released. Comparison
of amount of enzyme-released ammonia to total potential provided an estimate of
extent of deamidation as shown also in Table 5 .

Table 5 Estimation of extent of deamidation of insoluble wheat peptides

 0 0
60 3.7
90 8.2
120 10.9
180 14.6
240 17.3
300 20.2
360 2 1.7
420 23.0
500 24.5

Extent of deamidation determined by amino acid analysis

Confirmation of the extent of deamidation was achieved by amino acid analysis of the
untreated peptide preparation and the protein deamidase-treated preparation and
comparison of the sum of glutamic acid and aspartic acid content with the sum of
glutamine and asparagine contents in each preparation.

Example 3 . Soluble wheat peptides deamidated with protein deamidase show

enhanced emulsification capacity

Emulsification capacity of deamidated soluble wheat peptides was measured

according to a well-established procedure and compared to the emulsification capacity
of non-deamidated soluble wheat peptides and sodium caseinate, a proteinaceous
substance used widely in food emulsion systems owing to its excellent performance.

Method

A deamidated soluble wheat peptide sample ( extent of deamidation 30%), a non-

deamidated soluble wheat peptide sample or sodium caseinate was dispersed in water
at 50°C at a concentration of 0 .4 % w/w. The pH was adjusted to be in the range 7.0 to
7.2 by addition of dilute sodium hydroxide solution and stirred until fully dissolved.
Into a lOOOmL tall-form beaker, 125 mL of the test solution was placed and mixed
vigorously with a Silverson homogeniser. Into this, vegetable oil ( yellow in colour)
was added in a continuous stream, the quantity of oil being added was continuously
monitored gravimetrically. With continuous addition of oil the mix became creamy
white in colour and progressively more viscous as the oil-in-water emulsion being
formed included more oil. When the emulsification capacity of the sodium caseinate,
deamidated soluble wheat peptides or non-deamidated soluble wheat peptides was
exceeded, the emulsion transformed instantaneously into a water-in-oil emulsion
recognised by the sudden change in viscosity and colour change to yellow. At this
point the quantity of oil added was noted and used to calculate the Emulsification
Capacity (EC) expressed as weight of oil emulsified by l g emulsifier.

Results

Table 6 Emulsion capacities of deamidated soluble wheat peptides, non-deamidated

soluble wheat peptides and sodium caseinate

Emulsification capacity of deamidated soluble wheat peptides was demonstrated in a

coffee creamer emulsified food system according to an established procedure and
compared to the emulsification capacity of non-deamidated soluble wheat peptides
and sodium caseinate, a proteinaceous substance used widely in such food emulsion
systems owing to its excellent performance.

Method

Coffee creamer products were made with a deamidated soluble wheat peptide sample,
a non-deamidated soluble wheat peptide sample or sodium caseinate as the emulsifier.

Formulation

Glucose syrup (80% solids) 246g

Copha hydrogenated vegetable fat lOOg
Emulsifier 13g
Disodium hydrogen phosphate 6.5g
Water at 50°C 134g
Disodium hydrogen phosphate was dissolved in the hot water. The emulsifier was
added and dispersed fully using a Silverson homogeniser operating at 30% power.
Glucose syrup was added and dispersed. The Copha hydrogenated fat was melted at
50°C and slowly added while continuously homogenising the mix. When all the
ingredients were incorporated the homogenising power was increased to 100% and
continued for a further 2 minutes when the product appeared uniform.

Evaluation of coffee creamer emulsion product

Emulsion stability was evaluated by centrifugation of each of the coffee creamer

emulsions. 45g of each emulsion was dispensed into a 50mL capacity centrifuge tube
and centrifuged on an Heraeus benchtop centrifuge in a series of increasing
centrifugal severity. The centrifugation conditions and results are shown in Table 7 .

Table 7 Emulsion stability of coffee creamer emulsions evaluated by a centrifugation

test

Whereas the coffee creamer emulsions formulated with sodium casemate or

deamidated soluble wheat peptides maintained visually homogenous emulsions at all
levels of centrifugal severity employed, coffee creamer formulated with non-
deamidated soluble wheat peptides began to destabilise under the mildest centrifugal
conditions tested.

Emulsion stability of each of the coffee creamer emulsions was evaluated in a

standard coffee whitening test
Method

Black coffee was prepared by adding 5g of instant coffee granules into 200g boiling
water. When uniformly dissolved 5g of each of the coffee creamer emulsions was
added with gentle stirr ing into separate volumes of black coffee.

Results

Samples were evaluated visually over a period of time after standing with no further
stirring or heating.

Observations are provided in Table 8 .

Table 8 . Performance of coffee creamers emulsified with either deamidated soluble

wheat peptides, non-deamidated soluble wheat peptides or sodium caseinate.

Whereas the coffee creamer emulsions formulated with sodium caseinate or

deamidated soluble wheat peptides whitened black coffee solution satisfactorily,
coffee creamer formulated with non-deamidated soluble wheat peptides destabilised
immediately on addition to the hot coffee. All whitened coffee samples showed some
degree of destabilisation over 17 hours , the degree being very apparent as the coffee
cooled and the floating free fat solidified as a scum layer. However, the extent of
scum layer was very much greater in the sample containing non-deamidated soluble
wheat peptides as emulsifier relative to the extents in samples formulated with sodium
caseinate or deamidated soluble wheat peptides.

Example 4 Insoluble wheat peptides deamidated with protein deamidase

transformed into hydrated and gellable substance

Hydration of insoluble wheat peptides

The progress of deamidation of insoluble wheat peptides was shown in Example 2 ,
Table 5 . At intervals during the deamidation of insoluble wheat peptides samples were
withdrawn from the reaction mix and centrifuged to estimate possible change in
volume and appearance of insoluble material. Using graduated conical centrifuge
tubes the volume of sediment was read off directly and compared to that observed
from before reaction commenced.

Results and observations are shown in Table 9 . In the early stages of deamidation
reaction, the insoluble peptides apparently hydrated and expanded and a proportion
became dissolved or dispersed and were identified in the supernatant after
centrifugation by solids measurement and appearance. During the later stages of the
reaction no further expansion of the pellet was observed but further amounts of solids
appeared in the supernatant layer after centrifugation and a layer of lipid was noted on
the top of the liquid in the centrifuge tube.

Table 9 . Volume of sediment and appearance of s upernatant recorded during

deamidation of insoluble wheat peptides

Gelation of deamidated insoluble wheat peptides

Gelation of polymeric carbohydrates and proteins may induced by heating such

materials in aqueous solution or by adding cross-linking agents according to well-
established methods.. Both processes are deemed to bring about the formation of a
highly hydrated interwoven matrix of extended polymeric structures that entraps
water and forms a quasi-solid recognised as a gel.

Deamidated insoluble wheat peptides recovered after extensive deamidation ( 27%) as

aforesaid ( Table 10) were either heated in the aqueous dispersion to 95°C for 15 min
then cooled to ambient temperature or exposed to the sulphydryl oxidising and cross-
linking agent , dithiothreitol (0.5%) at 50°C or to both treatments, that is, after
addition of dithiothreitol, the sample containing deamidated insoluble wheat peptides
was heated at 95C for 15 min and then cooled to ambient temperature. The same
procedures were applied to non-deamidated insoluble wheat peptides

Results are described in Table 10 (a) and (b)

Table 0 Gelation of deamidated insoluble wheat peptides

(a) treatments applied to deamidated insoluble wheat peptides

Figure 1 Chromatogram showing HPLC analysis of soluble wheat peptides -
results for 3 x samples overlaid.

<' hn mat ni ni Suin n

It will thus be appreciated that this invention at least in the forms of the examples
described provides novel products derived from wheat gluten that can be used
advantageously as food ingredients in a range of foods additional to traditional uses
for gluten as in bakery goods, pasta and noodles. Additionally, processes are
disclosed for making the aforesaid novel products and applications for such novel
products in foods. The examples disclosed however are only the currently preferred
forms of the invention and additional modifications may be made within the scope of
the invention as defined by the following claims.
The claims defining the invention are as follows:

1. A method of produc ing an ingredient material for use in food, said method
including the steps of transforming a parent gluten complex by two or
more enzymatic processes whereby said ingredient material displays
different physical and functional properties to said parent gluten complex.

2 . The method as claimed in claim wherein said enzymatic processes occur

sequentially,

3 . The method as claimed in claim 1 wherein said enzymatic processes occur

concurrently.

4 . The method as claimed claim 1 wherein in one of said enzymatic

processes the parent gluten complex is fragmented by enzymatic
hydrolysis.

5 . The method as claimed in claim 4 wherein in another subsequent one of

said enzymatic processes fragmented gluten is changed by enzymatic

hydrolysis of a proportion of a glutamine constituent of peptides and
conversion thereof into glutamic acid.

6 . The method as claimed in claim 5 wherein said fragmented gluten is

separated according to size, charge or solubility

7 . The method as claimed in claim 1 wherein said enzymatic processes

include enzymatic proteolysis of a vital wheat gluten followed by
separation of soluble from insoluble peptides followed by enzymatic
deamidation of soluble peptides.
8 . The method as claimed in claim 6 wherein said fragmented gluten is dried

by any suitable means.

9 . The method as claimed in claim 7 wherein said fragmented gluten is dried

by spray drying.

10. The method as claimed in claim 1 wherein said parent gluten complex is a

vital wheat gluten.

11. The method as claimed in claim 9 wherein said one of said enzymatic
processes uses a commercial tryptic-like enzyme of fungal origin and said
another subsequent one of said enzymatic processes uses a protein-
deamidase enzyme.

12. A method of producing an ingredient material for food, said method being

substantially as described herein with reference to the examples.

13. A food product which includes the ingredient material produced by the

method as claimed in claim 1.

14. The food product as claimed in claim 12 wherein said food product is a

dough or batter in which the elasticity, extensibility and viscosity is

enhanced by said ingredient material.

15. The food product as claimed claim 12 wherein said food product is a meat

product or meat analogue product in which the inclusion of water and fat
components is enhanced by said ingredient material.

16. The food product as claimed in claim 12 wherein said food product is a

liquid food or beverage in which the inclusion of water and fat

components is enhanced by said ingredient material.
17. The food product as claimed in claim 1 wherein said food product is a

non-dairy fat powder or a non-dairy creamer in which emulsion stability is

enhanced by said ingredient material.
 INTERNATIONAL SEARCH REPORT International application No.
PCT/AU2015/000113

A . CLASSIFICATION OF SUBJECT MATTER

A23J 3/34 (2006.01) A23J 3/18 (2006.01) C12P 21/06 (2006.01) A23L 1/305 (2006.01) A23L 2/66 (2006.01)
A21D 2/26 (2006.01)

According to International Patent Classification (IPC) or to both national classification and IPC
B . FIELDS SEARCHED
Minimum documentation searched (classification system followed by classification symbols)

Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched

Electronic data base consulted during the international search (name of data base and, where practicable, search terms used)
EPODOC, WPIAP, SPATEN cluster, CAPLUS, FSTA, MEDLINE, Espacenet, Google Patents, Google Scholar: IPC/CPC marks
A23J, C12P21/06, C12P13/14, A23L1/228, A23L1/305, A23L2/66, A23K1/165, A21D2/24, A21D13/06 (in applicable databases);
Keywords (gluten, wheat protein, enzymolysis, protease, trypsin, deamidase, glutaminase, two-step, multiple step, food) and like
terms.

Espacenet: Applicant/Inventor name search.

C. DOCUMENTS CONSIDERED TO BE RELEVANT

Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to
claim No.

Documents are listed in the continuation o f Box C

X Further documents are listed in the continuation o f Box C X See patent family annex

* Special categories of cited documents:

"A" document defining the general state of the art which is not
considered to be of particular relevance
"E" earlier application or patent but published on or after the
international filing date
"L" document which may throw doubts on priority claim(s) or
which is cited to establish the publication date of another
citation or other special reason (as specified)
"O" document referring to an oral disclosure, use, exhibition
or other means
"P" document published prior to the international filing date
"T" later document published after the international filing date or priority date and not in
conflict with the application but cited to understand the principle or theory
underlying the invention
"X" document of particular relevance; the claimed invention cannot be considered novel
or cannot be considered to involve an inventive step when the document is taken
alone
"Y" document of particular relevance; the claimed invention cannot be considered to
involve an inventive step when the document is combined with one or more other
such documents, such combination being obvious to a person skilled in the art
"&" document member of the same patent family

Date of the actual completion of the international search Date of mailing of the international search report
2 April 201 5 08 April 201 5
Name and mailing address of the ISA/AU Authorised officer

AUSTRALIAN PATENT OFFICE Tien Ngo

PO BOX 200, WODEN ACT 2606, AUSTRALIA AUSTRALIAN PATENT OFFICE
Email address: pct@ipaustralia.gov.au (ISO 9001 Quality Certified Service)
Telephone No. +61 2 6283 2243

Form PCT/ISA/210 (fifth sheet) (July 2009)

 INTERNATIONAL SEARCH REPORT International application No.
C (Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT PCT/AU2015/000113

Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

LEE, S . E. et al., 'Effects of enzymatic modification of wheat protein on the formation

of pyrazines and other volatile components in the Maillard reaction', Food Chemistry
2012, Vol. 13 1, pages 1248-1254
X Abstract, sections 2 . 1, 2.2 and 2.4 at page 1249 1-1 1, 13

U S 2002/0106424 A l (OGAWA et al.) 08 August 2002

X Abstract, [0027], [0030], Examples 4 and 5 at [0044], [0048] 1-1 1, 13

WO 2002/001963 A2 (SOCIETE DES PRODUITS NESTLE S.A.) 10 January 2002

X Abstract, page 2 lines 1-5, page 4 lines 6-1 1, page 6 line 16 to page 7 line 28, Example 1-1 1, 13
1

US 2012/0288587 A l (CHENG et al.) 15 November 2012

X Abstract, [0001], [0021-0028], [0039-0042], Examples 1-2, Fig. 1 1-1 1, 13

SCHLICHTHERLE-CERNY, H. et al., 'Analysis of Taste-Active Compounds in an

Enzymatic Hydro lysate of Deamidated Wheat Gluten', J. Agric. Food Chem. 2002, Vol.
50, pages 15 15-1 522
X Abstract, Materials and Methods at page 15 16 1-1 1, 13

CN 1025245 17 A (BAODING WAY CHEIN FOOD INDUSTRIAL CO., LTD) 04

July 2012, Derwent DWPI Online Abstract Accession No. 2012-L42450
X Abstract 1-1 1, 13

WO 2001/076391 A l (SOCIETE DES PRODUITS NESTLE S.A.) 18 October 2001

X Abstract, page 4 line 17 to page 7 line 32, Examples 1-6 1-1 1, 13

Form PCT/ISA/210 (fifth sheet) (July 2009)

 INTERNATIONAL SEARCH REPORT International application No.
PCT/AU2015/000113

Box No. II Observations where certain claims were found unsearchable (Continuation of item 2 of first sheet)

This international search report has not been established in respect of certain claims under Article 17(2)(a) for the following
reasons:
1. I Claims Nos.:
because they relate to subject matter not required to be searched by this Authority, namely:
the subject matter listed in Rule 39 on which, under Article 17(2)(a)(i), an international search is not required to be
carried out, including

Claims Nos.: 12, 14-17

because they relate to parts of the international application that do not comply with the prescribed requirements to such
an extent that no meaningful international search can be carried out, specifically:
See Supplemental Box

3. I Claims Nos:
because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6 .4 (a)

Box No. Ill Observations where unity of invention is lacking (Continuation of item 3 of first sheet)

This International Searching Authority found multiple inventions in this international application, as follows:

As all required additional search fees were timely paid by the applicant, this international search report covers all
searchable claims.
I I As all searchable claims could be searched without effort justifying additional fees, this Authority did not invite
payment of additional fees.
I I As only some of the required additional search fees were timely paid by the applicant, this international search report
covers only those claims for which fees were paid, specifically claims Nos.:

No required additional search fees were timely paid by the applicant. Consequently, this international search report is
restricted to the invention first mentioned in the claims; it is covered by claims Nos.:

Remark on Protest | | The additional search fees were accompanied by the applicant's protest and, where applicable,
the payment of a protest fee.

I The additional search fees were accompanied by the applicant's protest but the applicable
protest fee was not paid within the time limit specified in the invitation.

I No protest accompanied the payment of additional search fees.

Form PCT/ISA/210 (third sheet) (July 2009)

 INTERNATIONAL SEARCH REPORT International application No.
PCT/AU2015/000113
Supplemental Box

Continuation of Box II
The claims do not comply with Rule 6.2(a) because they rely on references to the description and/or drawings.

Form PCT/ISA/210 (Supplemental Box) (July 2009)

 INTERNATIONAL SEARCH REPORT International application No.
Information on patent family members PCT/AU2015/000113
This Annex lists known patent family members relating to the patent documents cited in the above-mentioned international search
report. The Australian Patent Office is in no way liable for these particulars which are merely given for the purpose of information.

Patent Document/s Cited in Search Report Patent Family Member/s

Publication Number Publication Date Publication Number Publication Date

U S 2002/0 106424 A l 08 August 2002 JP 2002300862 A 15 Oct 2002

WO 2002/00 1 63 A2 1 January 2002 A 030298 A l 20 Aug 2003

AU 6593501 A 14 Jan 2002

PE 00732002 A l 07 Feb 2002

U S 2012/0288587 A l 15 November 2012 US 8828462 B2 09 Sep 2014

CA 2776669 A 1 12 Nov 2012

CN 102845710 A 02 Jan 2013

CN 1025245 17 A 04 July 2012

WO 2001/076391 A l 18 October 2001 AU 4422901 A 23 Oct 2001

BR 0109856 A 03 Jun 2003
BR 0109856 B l 29 Apr 2014
CA 2401961 A l 18 Oct 2001

CN 1419416 A 2 1 May 2003

EP 12743 18 A l 15 Jan 2003

EP 12743 18 B l 20 Jul 201 1

JP 2003529383 A 07 Oct 2003
KR 20030005268 A 17 Jan 2003

NZ 520886 A 30 Apr 2004

PL 35843 1 A l 09 Aug 2004
US 2003 1 13403 A l 19 Jun 2003

US 6838100 B2 04 Jan 2005

End of Annex

Due to data integration issues this family listing may not include 10 digit Australian applications filed since May 2001.
Form PCT/ISA/210 (Family Annex)(July 2009)