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Title:
PROCESS FOR THE PREPARATION OF SEMI-FINISHED FLOUR BASED FOOD PRODUCTS COMPRISING AN ELEMENT WITH TRANS-GLUTAMINAS ACTIVITY AND A SOURCE OF LYSINE
Document Type and Number:
WIPO Patent Application WO/2015/052665
Kind Code:
A1
Abstract:
A process for the preparation of semi-finished flour-based food, comprising the steps of providing a predetermined amount of at least one vegetable flour containing gluten; realizing a semi-finished dough product based on vegetable flour, adding the dough with an element with trans-glutaminase activities, adding at least one source of lysine adapted to cooperate with the element with trans-glutaminase activity for activating its chelating action of the QXP gluten antigenic sites by transamidation. The lysine source is a biological or a cell lysate, obtained by mechanical or physical treatments of natural products and added directly into the semi-finished dough.

Inventors:
CICIULLA, Danilo (Via Antonio Gramsci 49, Carlentini, I-96013, IT)
CORMACI, Gianfrancesco Emanuele (Via Filippo Corridoni 59, Mantova, I-46100, IT)
Application Number:
IB2014/065152
Publication Date:
April 16, 2015
Filing Date:
October 08, 2014
Export Citation:
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Assignee:
CICIULLA, Danilo (Via Antonio Gramsci 49, Carlentini, I-96013, IT)
CORMACI, Gianfrancesco Emanuele (Via Filippo Corridoni 59, Mantova, I-46100, IT)
International Classes:
A21D2/26; A21D8/04; A21D13/06
Domestic Patent References:
2010-02-11
1999-11-11
2008-05-08
1999-11-11
2008-05-08
Foreign References:
EP0963704A21999-12-15
EP0938845A11999-09-01
EP0948905A21999-10-13
US20100316764A12010-12-16
FR2973989A12012-10-19
US20110256268A12011-10-20
EP0262669A21988-04-06
US3650764A1972-03-21
GB2075054A1981-11-11
FR947040A1949-06-21
EP0963704A21999-12-15
EP0938845A11999-09-01
US20100316764A12010-12-16
US20020061344A12002-05-23
US5279839A1994-01-18
EP2548443A12013-01-23
Other References:
WATANABE M ET AL: "CONTROLLED ENZYMATIC TREATMENT OF WHEAT PROTEINS FOR PRODUCTION OF HYPOALLERGENIC FLOUR", BIOSCIENCE BIOTECHNOLOGY BIOCHEMISTRY, JAPAN SOCIETY FOR BIOSCIENCE, BIOTECHNOLOGY, AND AGROCHEMISTRY, TOKYO, JAPAN, vol. 58, no. 2, 1 February 1994 (1994-02-01), pages 388 - 390, XP000435777, ISSN: 0916-8451
Attorney, Agent or Firm:
MARINO, Ranieri (Contrà Paolo Lioy 24, Vicenza, I-36100, IT)
Download PDF:
Claims:
Claims

1. A process for the preparation of flour-based semi-finished food, comprising the following steps:

a) providing a predetermined amount of at least one vegetable flour containing gluten; b) realizing a semi-finished dough based on said at least one vegetable flour;

c) adding an element with trans-glutaminase activity to said dough;

d) adding at least one source of lysine adapted to cooperate with the element with trans-glutaminase activity to activate its chelating action of the QXP gluten antigenic sites by transamidation;

characterized in that said lysine source is a biological or cellular lysate obtained by mechanical or physical treatments of natural products and added directly into said semifinished dough.

2. Process according to claim 1, characterized in that said lysate is used as raw form or as supernatant and is derived by processing a microorganism containing lysine

3. Process according to claim 2, characterized in that said microorganism is selected into the group comprising Saccharomyces spp.; Schizosaccharomyces spp.;

Saccharomycopsis spp.; Lactobacillus spp.; Leuconostoc spp.; Pediococcus spp;

Bifidobacterium spp.; Ruminococcus spp.; Selenomonas spp.; Glucobacter spp.;

Chlamydomonas spp.; Chlorella spp.; Chlorobium spp.; Chlorococcum spp.; Spirulina spp.; Volvox spp.; Spyrogyra spp.; Cytophaga spp.; Rhodobacter spp.;

Rhodopseudomonas spp.; Rhodo spirillum spp.; Rhodomicrobium spp.; Desulfovibrium spp.; Flavobacterium spp.; Desulfuromonas spp.; Thiobacillus spp.; Paracoccus spp.;

Sorangium spp.; Rhizobium spp.; Agrobacterium spp.

4. Process according any preceding claims, characterized in that said microorganism is subjected to cold sonication to carry out the lysis of the fungal cell wall and generate a cell lysate.

5. Process according claim 4, characterized by comprising a step of cooling the lysate at a temperature below 0°C, preferably close to -20 °C, to prevent hydrolysis reactions, said cooling step being carried out until the lysate is added in the semi-finished product. 6. Process according to any of claims 1 to 3, characterized in that said source of lysine is subjected to a mechanical treatment of homogenization or centrifugation adapted to provide at least partially said element with trans-glutaminase activity.

7. Process according to any one of the preceding claims, characterized by comprising a step of addition in the supernatant of a source of papain, purified and crystallized as a single enzyme, adapted to hydrolyze the supernatant proteins derived from the mechanical treatment and to promote the reaction with the PNG-ase activity contained in the sediment.

8. Process according to any of the preceding claims, characterized by comprising a step of adding a source of at least one of the enzyme 1,3-beta-glucanase and Class II and III chitinase, to release the transglutaminase activity of the centrifuged cell wall fraction of said microorganism, in the form of fresh papaya juice or of any nature based on.

9. Process according to any one of the previous claims, characterized in that said lysine source is selected from components and elements containing and/or producing nisin, such as non-pathogenic strains of Streptococcus spp., Staphylococcus spp., Ruminococcus spp., Bacillus spp., Carnobacterium spp., Halobacterium spp., Actinoplanes spp., Kluyveromyces spp., Leuconostoc spp. and Lactobacillus spp.

10. Process according to any one of the preceding claims, characterized by comprising a step of adding to the lysate of a non-toxic reducing agents selected from the group comprising sorbic acid, benzoic acid, sodium metabisulfite, potassium metabisulfite, calcium metabisulfite, ascorbic acid, citric acid, tartaric acid, L-cysteine, L- cysteinehydrochloride, L-cysteine hydrochloride monohydrate, and similar agents adapted to reactivate the PNG-ase present in the cell wall of the microorganism.

11. Process according to any one of the preceding claims, characterized in that the TG- ase is at least partially derived from bacteria.

Description:
PROCESS FOR THE PREPARATION OF SEMI-FINISHED FLOUR BASED FOOD PRODUCTS COMPRISING AN ELEMENT WITH TRANS-GLUTAMINAS ACTIVITY AND A SOURCE OF LYSINE

Description

Technical Field

5 The present invention is generally applicable to the field of food treatment and

preparation and in particular relates with a new process for the preparation of semifinished flour-based food products.

State of the art

It is well known that gluten is a main component in most of food flours, and particularly

0 in the most widely used flours such as wheat flour, as the gluten during digestion is

processed by different enzymes allowing its proper hydrolysis. In the intestine, the presence of the enzyme trans-glutaminase (TG-ase) is crucial for the conjugation of gluten proteins with TG-ase itself, making antigenic the whole complex under proper conditions.

5 In particular, in persons with celiac disease the presence of gliadin, one of the gluten

proteins, triggers a pathological and inflammatory reaction, due to the onset of a specific antigen response.

That is why the food industry has tried to develop a range of food products specifically designed for celiac patients and therefore "gluten-free".

0 In particular, several products that require the replacement of flour with other gluten- containing vegetable flours have been formulated, such as corn flour, rice flour, buckwheat flour and the like, either devoid of genes encoding gluten proteins or with a minimum content as they can be considered gluten-free.

Other solutions provide, however, the formulation of products from bio-engineered

5 wheat flour lacking the expression of the gluten proteins.

This has meant that for the production of gluten-free food, special precautions were required for the design phase of production sites, supplying methods and machining processes.

In addition, the use of gluten-free or artificially deglutinated flours causes obvious

0 quality defects found in the finished products. In fact, the finished products classifiable

as gluten-free have a mediocre appearance and a poor palatability, with a resulting impaired life quality for people suffering from celiac disease.

The production of deglutinated flours, starting from genetically modified organisms to delete the gene pool responsible for the synthesis of gliadin and glutenin (major components of gluten), is extremely expensive and laborious.

The need for eliminating the gluten from standard flours also requires special precautions that need appropriate changes of the production plants, both in the design and in the building steps.

Not least, the lack of gluten and the consequent elimination of gluten network, that gives the right elasticity to the dough, impairs their rheological properties and workability, requiring complex processing steps from the technical point of view.

As a consequence, in addition to being poor in flavour and from an organoleptic point of view, the finished products are also relatively expensive.

To overcome such drawbacks, some solutions have been provided where it was tried to neutralize the gluten using a bacterial trans-glutaminase (TG-ase), along with a laboratory enzymatic cofactor containing lysine (K), with the purpose of chelating gluten protein antigenic sites, precisely the QXP sequences responsible to trigger the antigenic response in celiac subjects.

In particular, it is known the use of a chemical reagent called lysine-methyl-ester (K- Me), a synthetic product with a relatively high cost, has allowed to obtain acceptable but economically unfavourable results.

The use of trans-glutaminase is also known in the food industry, in combination with regular gluten flours, with the ultimate purpose of improving the rheology and technological properties of dough, to perform enrichment in protein content and possibly an overall organoleptic improvement.

Examples of such solutions are disclosed in EP0963704, EP0938845, US2010316764, US2002061344, US 5279839 and EP 2548443.

However, the use of TG-ase in the absence of a lysine source is acknowledged not to produce the gluten chelation and, therefore, the solutions mentioned above are not suitable for the production of foods addressable for people either with celiac disease or any other gluten-related disorder.

From WO99056698, instead, a method for the treatment of celiac disease is known which involves the use of lysine, a derivative thereof or other simple amines such as methylamine, ethylamine, putrescine, spermidine and spermine as TG-ase co-substrates, for the molecular sequestration of specific portions of the gliadin and possibly for the glutenin, for the deamidation of a glutamine residue in specific sequences of the gluten, since this document argues, although it is not uniquely determined, that just a deamidation of specific glutamines would exclusively trigger the immune response of celiac patients.

However, to date, is unknown if the antigenic response to gluten may be also mediated from other gliadin sequences, in addition to the known ones, so that this solution does not allow to inactivate all the QXP sequences present in the gliadin and cancel any risk associated with the ingestion of gluten, but focuses exclusively on certainly known celiacogenic sequences.

Not least, the described process is implemented by acting directly on flour and not on intermediates of the production, i.e. on the dough, with resulting effects in terms of higher costs for the final product.

In turn, WO2008053310 describes a method of treatment of flour-based food and their use by celiac subjects, which involves the use of TG-ase as a gluten masking catalyst, also in this case by acting directly on flour but through transamidation.

The substrate masking gluten is a derivative of lysine, precisely an ester of this aminoacid with a primary alcohol in one, two, three or four carbon atoms or, possibly, a lysine peptide derivative or another primary amine and therefore always through relatively complex chemical treatments, highlighting as a result the aforementioned technological problems and costs.

Also in this case, then, the process focuses on flour and not directly on the semi-finished intermediate.

Scope of the invention

The object of the invention is to overcome the above drawbacks, providing a process for the preparation of semi-finished flour-based food which is particularly efficient and relatively cheap.

A particular object is to provide a process for the preparation of semi-finished flour- based foods which allows to obtain products with a low content of gluten or labelled gluten-free, using any vegetable flour containing gluten and avoiding the use of flours bio-engineered for not expressing the gluten.

A particular object is to provide a process for the preparation of semi-finished flour- based foods which allows to obtain products with a low content of gluten or labelled gluten-free, by acting directly in the mixing phase of the raw materials and without the use of special flours. A further object is to provide a process for the preparation of semi-finished flour-based foods which allows to obtain products with a low content of gluten or classifiable as gluten-free, using natural components already employed in oven -baked goods.

Still another particular object is to provide a process for the preparation of semi-finished flour-based foods which allows to obtain products with a low content of gluten or classifiable as gluten-free intended to cancel either the causes that generate the celiac disease and the risk associated with gluten ingestion.

Another particular object is to provide a process for the preparation of semi-finished flour-based foods which allows to obtain products with a low content of gluten or labelled gluten-free which allow to have products matching to traditional products both for organoleptic and rheological properties and with regard to flavour and taste.

Not last object of the invention is to provide a process for the preparation of semifinished flour-based foods which allows to obtain products with a low content of gluten or labelled gluten-free, and which can be implemented in traditional industrial systems, without making substantial modifications thereto, but only with the insertion of simple to build and easy-to-use auxiliary equipments.

These objects, along with others that will appear more clear hereinafter, are achieved thanks to a process for the preparation of semi-finished flour-based food which, in accordance with claim 1, comprises the steps of providing a predetermined amount of at least one vegetable flour containing gluten, realizing a semi-finished dough product based on said at least one vegetable flour, adding to said dough an element with transglutaminase activity, adding at least one source of lysine adapted to cooperate with the element with trans-glutaminase activity to activate its chelating action of the gluten QXP antigenic sites by trans-amidation, wherein said lysine source is a biological or cellular lysate obtained by mechanical or physical treatments of natural products and added directly into said semi-finished dough.

Thanks to this features combination, the contribution of lysine needed to trigger the TGase-dependent transamidation will be directly performed during the mixing of the raw materials, without the need of any bio-engineered flour.

Consequently, it could be possible to use readily available and hence more economic raw materials, which maintain unaltered the rheological properties, in order to allow the workability of the semi-finished product.

As matter of fact, the flour gluten network will not be altered, then the dough obtained from working said semi-finished product will show optimal elasticity and strength, suitable to allow the processing of semi-finished product in traditional plants.

Suitably, said lysate may be used in raw form or as a supernatant and derived from the treatment of a microorganism containing lysine, such as a microorganism selected from Saccharomyces spp.; Schizosaccharomyces spp.; Saccharomycopsis spp.; Lactobacillus spp.; Leuconostoc spp.; Pediococcus spp.; Bifidobacterium spp.; Ruminococcus spp.; Selenomonas spp.; Glucobacter spp.; Chlamydomonas spp.; Chlorella spp.; Chlorobium spp.; Chlorococcum spp.; Spirulina spp.; Volvox spp.; Spyrogyra spp.; Cytophaga spp.; Rhodobacter spp.; Rhodopseudomonas spp.; Rhodo spirillum spp.; Rhodomicrobium spp.; Desulfovibrium spp.; Flavobacterium spp.; Desulfuromonas spp.; Thiobacillus spp.; Paracoccus spp.; Sorangium spp.; Rhizobium spp.; Agrobacterium spp.

In this way it will be possible to use natural products suitably treated by physical and/or chemical processes, some of which may already be present in the commonly employed ingredients of baked goods, such as S. Cerevisiae of the Saccharomyces family.

Advantageously, the microorganisms or other lysine sources may be subjected to a cold sonication or other mechanical treatments, such as homogenization or centrifugation, that do not require any modification of plants but the addition of simple and easy-to-use auxiliary equipments, in such a manner to not significantly increase the overall costs. Also, the use of mechanical treatments such as homogenization or centrifugation will allow to at least partially provide the element with trans-glutaminase activity, also avoiding the use of further bacterial TG-ase in the mixing phase.

Advantageous embodiments of the process are obtained according to the dependent claims.

Brief description of the drawings

Further features and advantages of the process according to the invention will become more apparent in light of the detailed description of some preferred but non exclusive embodiments of a process according to the invention, illustrated in way of non limiting examples with the aid of the accompanying drawings, wherein:

FIG. 1 is a block diagram of a first preferred embodiment of the process;

FIG. 2 is a block diagram of a second preferred form of execution of the process.

Detailed description of some preferred embodiments

With regard to the above figures, a process for the preparation of flour-based semifinished foods is illustrated, which semi-finished product being suitably machined in one or more further successive steps to obtain baked or other food products such as pasta or the like, with no particular limitations.

The final treatment of the semi-finished product, as portioning, cooking, storing, packaging, deep-freezing and other steps necessary to obtain a product suitable for consumption are not described as not limiting the scope of protection of the present invention.

In Fig. 1 a first mode of carrying out the invention is showed, which essentially comprises a step a) of providing a predetermined amount of a vegetable flour containing gluten or a mixture of vegetable flours, at least one of which contains gluten, in variable proportions and amounts depending on the recipe.

Further ingredients are subsequently added (step ao) to flour or flour mixture, and in particular one or more liquid phases, in order to achieve a semi-finished dough (step b). Also in this case, of course, the choice of the other components may be highly dependent on the desired finished product.

The mixture will then be added (step c) with a trans-glutaminase activity element, which could be directly added to the mixture or introduced together with the other ingredients. The trans-glutaminase element may be of bacterial origin.

The dough is then added (step d) with at least one source of lysine adapted to cooperate with the element with TG-ase activity to activate its chelating action on the gluten QXP antigenic sites by trans-amidation, thus obtaining a food product suitable for subjects with celiac disease or other gluten-related disease.

The source of lysine, or one or more of the provided lysine sources, will be a biological or cellular lysate obtained by mechanical or physical treatments of natural products and added directly into the semi-finished dough.

In particular, the lysate may be used in raw form or as a supernatant resulting from treatment of a microorganism containing lysine, such as yeasts or chlorophyceae (green microalgae), with selection of strains considered non-pathogenic to man.

For example, the microorganism will be selected among the non-pathogenic strains of Saccharomyces spp., Schizosaccharomyces spp., Saccharomycopsis spp., Lactobacillus spp., Leuconostoc spp., Pediococcus spp., Bifidobacterium spp., Ruminococcus spp., Selenomonas spp., Glucobacter spp., Chlamydomonas spp., Chlorella spp., Chlorobium spp., Chlorococcum spp., Spirulina spp., Volvox spp., Spyrogyra spp., Cytophaga spp., Pvhodobacter spp., Rhodopseudomonas spp., Rhodo spirillum spp., Rhodomicrobium spp. Desulfovibrium spp., Flavobacterium spp., Desulfuromonas spp., Thiobacillus spp., Paracoccus spp., Sorangium spp., Rhizobium spp., Agrobacterium spp.

In a particular application, the lysine source or one of the sources of lysine may be milk, in any form, added directly to the dough.

According to a further variant, the lysine source or one of the sources of lysine may be selected among elements containing and/or producing nisin, such as non-pathogenic strains of Streptococcus spp., Staphylococcus spp., Ruminococcus spp., Bacillus spp., Carnobacterium spp., Halobacterium spp., Actinoplanes spp., Kluyveromyces spp., Leuconostoc spp. and Lactobacillus spp.

In the example of Fig. 1, the suitably prepared micro-organism (step eo) is subjected to cold sonication treatment (step e), adapted to achieve the lysis of the fungal cell wall for generating a cell lysate which make available the lysine necessary for the TG-ase to mask the gluten QXP antigenic sites.

The lysate is obtained by the action of ultrasounds on regular baker's yeast, that can be immersed in a basin of cold water, which acts as a cooling medium, and treated for 2.5 minutes with ultrasonic frequencies.

The step e) of sonication is followed by a step f) of centrifugation necessary to separate (step g) the supernatant from the solid sediment, that can be separately collected (step h) and possibly removed (step i), while the supernatant will be introduced in the dough (step d).

The complex of the steps of mechanical treatment of the selected microorganism may be performed in parallel to the step of preparation of the dough, or before the same. In the latter case it will be appropriate to provide a cooling phase of the lysate below 0°C, preferably close to -20°C, to prevent hydrolysis reactions and the lysate will be maintained cooled until its addition in the semi-finished product or dough.

Before adding the dough with a lysine source, dough itself can be added (step j) with further adjuvants like a source of papain, purified and crystallized as a single enzyme or as a recombinant enzyme, to take advantage of its transamidase ability by pH variation of the dough in the range of values between 5.7 and 7.5 and in particular of its ability to hydrolyze the proteins in the supernatant derived from the mechanical treatment, to enhance the reaction with transglutaminase contained in the solid sediment.

A further additive may be made up from at least one source of the enzyme 1,3-beta- glucanase, and the Class II and III chitinases, for example fresh papaya juice or any papaya-based sample or compound, in order to release the PNG-ase within the microorganism cell wall fraction, through degradation of the cell wall polysaccharides themselves exerted by these enzymes.

Additional additives to the lysate may be non-toxic reducing agents selected from the group comprising sorbic acid, benzoic acid, sodium metabisulfite, potassium metabisulfite, calcium metabisulfite, ascorbic acid, citric acid, tartaric acid, L-cysteine, L-cysteine hydrochloride, L-cysteine hydrochloride monohydrate, and similar agents to reactivate the cell wall PNG-ase of the microorganism.

The dough so obtained will undergo one or more subsequent accessory steps, such as a resting step k) for a predetermined time, e.g. 60 minutes, a step 1) of new kneading with the possible addition of yeast (phase lo), a possible step m) of leavening in the case where the semi-finished product is intended for the production of bakery products, a portioning step n), and further steps o) of cooking or other storage procedure designed to obtain the product P ready for consumption and/or sale.

Fig. 2 shows a second mode of carrying out the process, which differs from the previous essentially because the step e) of the lysine source sonication is replaced by a mechanical treatment of centrifugation, for example using a piston-rod mechanical homogenizer, in order to release either the lysine useful to the enzymatic reaction and the cell wall with PNG-ase activity, to replace the external supplementation of bacterial TG-ase or as partial addition to the external Tg-ase amount itself.

Treating the supernatant with papain will hydrolyze its protein content, making more easily available for the reaction with TG-ase contained in the sediment, which therefore will also be suitably introduced into the dough.

From above it is evident that the process according to the invention reaches the intended objects.

The above disclosed process is susceptible of numerous modifications and variations. All the details may be replaced with other technically equivalent elements, and the materials may be different according to requirements, without departing from the scope of the present invention.

Even if the process has been disclosed with particular reference to the attached figures, reference numbers used in the description and in the claims are used to improve the intelligence of the invention and do not constitute any limitation the claimed scope.