WO2014121363A1

WO2014121363A1 - Gluten-free beer production process

Info

Publication number: WO2014121363A1
Application number: PCT/BR2014/000041
Authority: WO
Inventors: Paulo VEIT
Original assignee: Veit Paulo
Priority date: 2013-02-08
Filing date: 2014-02-06
Publication date: 2014-08-14
Also published as: BR102013003182B1; BR102013003182A2; BR102013003182A8

Abstract

A process is disclosed for producing gluten-free beer from grains and raw materials containing this protein, by causing the beer to undergo during its production a complete protease process with gluten degrading enzymes, the gluten being subsequently removed from the beer, allowing the beer to be consumed by celiac disease patients; in addition, during the fermentation phase, yeasts multiplied in a gluten-free medium are used and allow the enzymes used during processing to be re-used.

Description

"GLUTEN-FREE BEER MANUFACTURING PROCESS"

The present patent relates to a gluten-free brewing process which is made from grains and raw materials that have this protein, and during its production is subjected to a proteasf process with degrading enzymes. Gluten, which will later be removed from beer, allowing its consumption by people with celiac disease, and employing yeasts that were multiplied in gluten-free medium during its fermentation phase.

Drinking is a habit among people, but a portion of the world's population has an autoimmune condition caused by gluten intolerance, which affects the small intestine and is called celiac disease.

Celiac disease causes a number of complications for its sufferers, such as diarrhea, growth difficulties, fatigue, weight loss, and other diseases such as anemia, osteoporosis, lymphoma, and bowel ulcers.

She has no treatment or vaccine and celiac patients need to refrain from consuming products containing the gluten protein to have a near-normal life.

Usually the beers are produced with raw materials, malted or unmalted, containing gluten such as barley, rye, wheat and oats, which restricts the consumption of this type of drink by celiac.

In order to supply this consumer market, options have been developed for gluten-free beers, using mostly raw materials that do not have this protein and, more recently, research has begun to genetically modify the beans, so that they do not have gluten protein. One of the prior art cases is represented by patent ZA 9902543 which uses maize or sorghum as a raw material and submits these materials to a saccharification process using amylolytic enzymes and glucanase to obtain a gluten free beer for Celiac people. The use of enzymes in this case is only for the saccharification of the must, since the grains used in the production are already gluten free.

Another example is patent application WO 2007/102850 which employs rice and sorghum and an enzymatic reaction to obtain the saccharification of these gluten-free materials to make a gluten-free beer,

US 2005/0170042 works with honey, sugar molasses, hops, moss / lichen and yeast to make gluten free beer.

German patent DE 102006033232 uses cassava, maize, rice and potatoes as a raw material and adds calcium sulfate and hops to obtain a gluten free beer.

Another German patent DE 102005020639 employs gluten free grains such as corn, buckwheat, quinoa or amaranth to make a gluten free beer.

Patent application WO 2012105977 also starts from quinoa grains to generate a gluten free beer.

There is patent application WO 2008009096 which uses concentrated pea, soybean, rice or potato proteins in place of malt proteins to make a beer consumed by celiac people.

WO 2006/051093 discloses the process for obtaining a low gluten drink using transglutaminase, peroxidase, hexose oxidase, tyrosinase, lacase or disulfide isomerase enzymes which interact with the gluten protein to form protein aggregates. that are not identified by the organism of the person with celiac disease. Through this transglutaminase process, gluten binds to other proteins and the formed molecule aggregate is not recognized. by the celiac organism. In unscientific language, although present in the drink, gluten is encapsulated by other proteins, forming a component that is no longer recognized by the celiac organism.

There are also scientific studies and researches seeking the production of genetically modified wheat, oats, barley, rye and malt to not have gluten protein.

While these patents and patent applications have been a major advance in human knowledge, the current state of the art has some disadvantages,

The use of raw materials that do not have gluten protein has disadvantages because it alters several characteristics of beer, including its taste, color, taste, foam and beer density.

Thus, the celiac consumer can drink a beer made from gluten-free grains, but cannot appreciate the usual characteristics of a beer made from wheat, barley, rye or oats, whether malted or not, which generates frustration and reinforces the feeling of food restriction due to the disease.

Another disadvantage concerns the supply of raw materials because these gluten free grains are usually employed in the production of other foods and their offer for beverage manufacture is more limited.

Considering the beers as an example, wheat, barley, rye and oat grains, malted or not, have part of their production normally directed to the manufacture of this type of beverage, being possible to establish supply contracts and schedule the production of beer. When we work with rice, buckwheat, millet, sorghum, quinoa, spelled and other gluten-free grains, the supply of these raw materials for brewing is not abundant as they are used in the production of other which makes it difficult for large-scale production and scheduling to produce beers made from gluten-free grains.

Another disadvantage is the possible cross contamination of the drink with gluten or gluten traces, which may trigger the autoimmune reaction in the celiac person.

World standards state that a product is gluten free if it has less than 20 ppm of this protein, ie 20 grams in 1 tonne.

To ensure this pattern, it is necessary to cultivate the gluten-free grains in soil in which no other plant that has gluten was cultivated in the last 3 seasons, besides the harvest and storage require equipment exclusively directed to the handling of these gluten-free grains.

Under the usual conditions of agricultural production, dedicating a planting area to gluten-free grains only or reserving equipment exclusively for harvesting and storing these types of grains is very costly for farmers and the possibility of this occurring is very low.

Another example of cross-contamination is associated with yeast used in the fermentation of beer and other beverages, as they are often multiplied in gluten-containing media, such as barley malt must. Thus, they become impregnated with gluten or with gluten traces, which can trigger the autoimmune reaction in celiac.

We can also mention the existence of variations between the laws of each country, which are not uniform in relation to the possible amount of gluten in a food to be considered gluten free, which leaves celiac always worried about whether or not consume a particular product. For example, countries like England and Spain state that a beer is gluten free if it has a gliadin content below 10 ppm, while most countries set this limit at 20 ppm. In order to overcome the disadvantages of the current state of the art, the present invention has been developed in a new, original and creative way to manufacture gluten-free beers from grains containing this protein by subjecting the material to complete protease by enzymes which provide degrade gluten, allowing its total removal of the drink, and the reuse of enzymes used in the process. The developed invention also employs, in the fermentation phase of the beverage, yeasts that have been multiplied in gluten-free medium, avoiding cross or residual contamination of the treated beverage.

The inventive process goes beyond current knowledge and is broadly applicable to both alcoholic and non-alcoholic beers, beer bases, beer concentrates and additives for grains and gluten raw materials.

The grains used in the manufacture of the drink can be any that have gluten, especially: wheat, barley, rye, oats and their variants or subspecies, regardless of whether they are malted or not.

By means of the invented invention, beer, its base or concentrate is brought into contact with enzymes of pepsin (EC 3.4.23.1 and EC 3.4.23.2), trypsin (EC 3.4.21.4), chymotrypsin ( Papain (EC 3.4.22.32.33), bromelain (EC 3.4.22.28 to 33), chymosin (EC 3.4.23.4), ficin (EC 3.4.22.3), family enzymes EC 3.4, with emphasis on the enzymes of the mucopepsin subclasses (EC 3.4.23.23), carboxypeptidase A (EC 3.4.17.1), carboxypeptidase B (EC 3.4.17.2) or glyoxa carboxypeptidase (EC 3.4.17.4).

Pepsin A (EC3.4.23.1), Pepsin B (EC3.4.23.2), Chymosin (EC 3.4.23.4) and Mucopepsin (EC 3.4.23.23) are enzymes belonging to the Al peptidase family containing endopeptidases. , which are mostly activated at acidic pH. (Biochemical Journal 290: 205-218 (1993))

The Al family contains peptidases which are commonly known as "acidic proteinases" or "carboxylic proteinases". The typical peptidase is pepsin A, which has been known for its contribution to the proteolysis of vertebrate stomach food proteins.

Centrocatalytic is formed by two asparagine residues that activate a water molecule, mediating the nucleophilic attack on the peptide bond. Most Al-family peptidases are active at acid pH, but some like renin are also active at neutral pH. For both cases, cofactors are not required (eg divalent cations).

Papain (EC 3.4.22.32.33), bromelain (EC 3.4.22.28 to 33), phycin (EC 3.4.22.3) are Cl family peptidases. They contain many endopeptities and some exopeptidases. The proteolytic activity of papaya latex has been known since 1880 (e.g. Martin, 1884). The active ingredient in papaya latex is papain and a large family of peptidases that are homologous to papain have now been recognized as ficin and bromelain. Catalytic residues of the Cl family have been identified as Cys (Cysteine) and His (Histidine), forming a sitiocatalytic.

Trypsin (EC 3.4.21.4), chymotrypsin (EC 3.4.21.1) are serine endopedtidases of the SI family. The SI family is the largest of the peptidase families, by the number of proteins that have been sequenced and by the number of peptidases with distinct activities (Biochemical Journal 290: 205-218 (1993)). The SI family peptidases contain a catalytic triad of His (histidine), asp (asparagine) and Ser (serine).

Carboxypeptidase A (EC 3.4.17.1), carboxypeptidase B (EC 3.4.17.2), gly-xaacarboxipeptidase (EC 3.4.17.4) are M14 family peptidases and contain metallocarboxipeptidases. Carboxypeptides and pancreatic A were discovered in approximately 1929 (Waldschmidt-Leitz & Purr, 1929). Zinc ion is coordinated in the site by 2 histidines and glutamate as well as a water molecule. The mechanism of regulation of carboxypeptidase activity by their peptides has been discussed in conjunction with their structures (eg Guaschet aL, 1992). (Biochemical Journal 290: 205-218 (1993)). Most peptidases in the family are carboxypeptidases, hydrolyzing only the C-terminal amino acid of polypeptide chains. These enzymes have a site that recognizes the free C-terminal group, which is the key determinant of specificity. Carboxypeptidase recognizes aromatic side chain residues and carboxypeptidase B prefers basic amino acids.

Enzymes will act on protein peptide bonds, disrupting or hydrolyzing these bonds, resulting in the breakdown of gluten and the formation of peptide fragments that are not recognized by the celiac organism.

Enzymes from one class or one subclass can be chosen as well as enzymes from two or more classes or subclasses can be used together, and this choice will depend on the type of beverage, the beans used in its manufacture and the characteristics of its production process, such as temperature, pH, process time and chemical agents added to the beverage (antioxidants, preservatives, acidulants, stabilizers, emulsifiers, among others).

Protease enzymes are placed in one or more containers containing a substrate or support such that they adhere to this substrate or support and the beverage circulates through this container, which is immersed in the tank with the beverage.

Preferably, this substrate or support will be calcium alginate or chitosan, but may also be acid cellulose fiber, gelatin, aga, polyacrylamide, chitin and alumina.

One or more containers containing the support and enzymes are placed in the treatment tank, which may be a tank already used in the usual brewing process or may be an exclusive gluten degradation tank. The location and attachment of the containers inside the treatment tank may vary at the discretion of the person performing the treatment.

The technical requirement is that the tank be equipped with pumps that ensure the circulation of the beer and its contact with the treatment enzymes.

During protease, temperature and pH must be constantly monitored because they interfere with the performance and effectiveness of gluten degradation, and in addition each enzyme type requires specific temperature and pH to perform better.

The duration, temperature and pH of the process will depend on the type of material to be treated, its quantity and the class or subclass of the enzymes used.

Normally, higher temperatures are more likely to accelerate the process of gluten degradation, but the allowable temperature limit of each enzyme cannot be exceeded so as not to cause its denaturation and affect the effectiveness of the gluten degradation process.

Treatment time lasts between 30 minutes and 10 hours, and preferably complete protease occurs between 4.0 and 6.5 hours.

The process temperature may range from 10 ° C to 90 ° C, but is preferably indicated from 40 ° C to 60 ° C.

The pH ranges between 3.0 and 10.0, preferably between

5.5 and 6.5.

After the stipulated treatment time for a given beer load, the gluten concentration in the beverage is measured and the material circulation is maintained until gluten is no longer identified.

This measurement uses the Elisa test, which evaluates the amount of gliadin, hordenine, secaline or avenin contained in the mixture. The developed process will be done until the Elisa test indicates values below 20 ppm, being preferable and possible to obtain values below 2 ppm residual gluten in the drink. It is noteworthy that the Elisa test for measuring gliadin, hordein, secaline and avenin failed to measure values less than 1.5 ppm for glidine and 3.0 ppm for hordein.

Internationally, for a drink to be considered gluten free, it must have less than 20 ppm gliadin, but the invented process allows values below 2 ppm gliadin to be achieved in the treated beer.

It has been observed that peptide fragments resulting from gluten degradation can randomly reorganize to form polymers that lack gluten characteristics and are not identified by the organism of the person with celiac disease.

Due to its characteristics and versatility, the invented process is easily inserted into the usual brewing processes.

Thus, the production processes of these beverages have a new stage, which is the phase of gluten degradation, with no changes in equipment or in the usual stages of production of these beverages, facilitating the use of the invention in the industry.

In summary, the invented process of gluten degradation will be introduced throughout the usual brewing process and will make it possible to obtain a gluten free drink that can normally be consumed by celiac people.

In addition to degrading gluten, it is important to note that the invented process also allows the recovery and reuse of enzymes employed in the treatment of the drink.

This is possible because most enzymes remain adhered to the support chosen for the treatment, ie the enzymes participate in the treatment of gluten degradation and remain in the container with the support.

Thus, simply remove the container containing enzymes and support and reuse it in another process. To further ensure gluten free in treated beer, the invention also acts in its fermentation phase so that the yeasts employed in this phase are multiplied in gluten-free medium.

In order to achieve this purpose, yeast strains suitable for each type of beer are multiplied in glucose medium or pre-treated medium to not contain gluten.

Treatment of the yeast multiplication medium employs the concept of the complete protease described in this report and uses the same classes and subclasses of enzymes presented in this invention. By way of example, a malt-based culture medium may be employed in yeast multiplication after undergoing the complete protease process described in this patent.

Thus, gluten-free beer obtained in the degradation phase of this protein will be fermented by yeasts that also do not have gluten or gluten traces, ruling out the chance of cross or residual contamination.

In order to facilitate understanding of the invention presented in this report, the following drawings and schemes are presented.

Figure 1 shows the treatment tank (1) in which the beer to be treated (2), the pump (3) and the pipes (4) allowing the circulation of the beer to be treated through the containers (5) is present. ), which contain the support or substrate (6) with the treatment enzymes (7).

Figure 2 shows a schematic flowchart of the brewing process pertaining to the current state of the art.

Figure 3 shows a schematic flowchart of the gluten-free beer production process with the introduction of the gluten degradation process disclosed in this patent and the use of multiplied yeasts in gluten-free medium.

Seeking a better presentation of the inventive concept and aiming to situate its participation in the production of beers, we will present briefly the stages of the brewing process belonging to the current state of the art and then we will report on the action of the invented process and locate the points of its action.

It is noteworthy that the manufacturing processes belonging to the current state of the art make it possible to obtain gluten beers.

In addition, it is also relevant to emphasize that it will be the invented process that will be responsible for obtaining gluten-free beers made from grains and raw materials that have this protein.

The current state of the art for the production of gluten beers begins with the milling of grains, continues with the saccharification or mashing stage, in which the malted grains are infused in hot water to turn their starch into fermentable sugars, and continue with the filtration step to remove grains deposited at the bottom of the processing tank.

After this filtration, the liquid goes through the boiling step, the addition of hops and cooling to a temperature of 20 ° C, and then sent to the fermentation tank, where it will be for a period of 5 to 10 days under controlled temperature between 12 ° C and 20 ° C.

Upon completion of fermentation, the must undergoes a precipitation step for sedimentation of the yeast, then cooled to 0 ° C and transferred to the maturation tank, where it will remain for approximately 10 days at - 1 ° C. The next step is to conduct the wort for the gasifying tank, which occurs the addition of C0 ₂ resulting in beer with gluten.

For purposes of this report, the beer wort is the speech during its manufacturing process before the addition of C0 _2.

The inventive process described in this patent will act prior to the wort boiling step and will enable wort gluten degradation, resulting in a beer totally free of this protein. By the invention described in this patent, the must will be subjected to complete protease by enzymes of the classes and subclasses of pepsin, trypsin, chymotrypsin, papain, bromelain, chymosin, phycin, enzymes of mucopepsin subclasses. carboxypeptidase A, carboxypeptidase B or gly-xaa carboxypeptidase, depending on the type of beer and grain used.

These enzymes will disrupt the peptide bonds of gluten molecules, resulting in the breakdown of this protein and the formation of peptide fragments that are not recognized by the celiac organism,

Exposure of must to enzymes for protease to occur is carried out in a tank with bombs that will circulate the must until all the gluten is degraded.

The same tank in which the must must boil may be used, provided it has pumps that enable the must to circulate, or we may use a specific tank for the degradation of gluten.

Enzymes are placed in one or more containers containing a support so that they adhere to this support and the must circulates through this container.

The support will be calcium alginate or chitosan, but acid cellulose fiber, gelatin, aga, polyacrylamide, chitin and alumina may also be used.

The processing time will depend on the amount of material to be treated and the amount and class of enzymes used, ranging from 30 minutes to 10 hours, and preferably complete protease occurs between 5.5 and 6.5 hours.

The pH will range between 3.0 and 10.0, preferably between

5.5 and 6.5. It is important to control the temperature and pH of the process and to measure the level of gluten concentration, maintaining the must circulation until gluten is no longer identified or until the gliadin concentration in the must is between 20 ppm and 0 ppm, and it is desirable for the gliadin level to be below 2 ppm.

The removal of peptide fragments resulting from gluten degradation will occur during the wort ripening stage through precipitation and filtration.

The invented process will also play an important role in the beer fermentation phase.

As fermentation uses yeast, it is essential that these yeasts be gluten free to avoid cross-contamination or residual must.

To ensure that the yeasts are gluten free, they are multiplied in glucose medium or in medium that has already undergone complete protease, such as malt medium that has undergone the gluten degradation step.

In tests conducted during the development of the present invention, pilsen beer made from barley grains was treated with trypsin, chymosin and chymotrypsin class enzymes for 6.0 hours at a temperature of 55 ° C and pH controlled at 6.2, giving a level of 1.5 ppm gliadin and 3 ppm hordein.

In these tests, the yeasts used in the fermentation phase were multiplied in gluten-free malted barley medium to ensure their gluten free or gluten free traces.

Gluten-free malted barley had previously undergone complete protease for degradation of its gluten.

In testing for gluten-free tripel beer production, gluten degradation used a combination of trypsin, chymosin and chymotrypsin, the degradation process time lasted 7.0 hours, at 51 ° C and pH controlled at 6.5. The measurements resulted in gliadin levels of 1.5 ppm and hordein of 3.0 ppm. Because the tripel beer required a much larger amount of grain in its production, it was necessary to employ a greater amount of enzymes in the process of gluten degradation.

To produce a gluten-free stout beer, we use enzymes from the trypsin, chymosin, chymotrypsin and carboxypeptidase classes. The treatment temperature was 55 ° C and the pH was controlled at 6.0. However, the duration of complete gluten protease lasted 7.5 hours, because in the production of this type of beer there is a greater amount of roasted malt grains, which makes the protease slower.

From the foregoing, it is apparent that the present invention solves the problems existing in the state of the art and contributes to the expansion of this field of human knowledge by presenting a novel and original way of producing a gluten-free beer from grains. and raw materials that have this protein, besides allowing the reuse of enzymes used in the complete protease of gluten proteins.

One of the advantages of the invented process is that it combines performance independence and ease of application with processes pertaining to the state of the art.

This is because the invented process performs the degradation of gluten present in the beer wort independently but is easily coupled with existing brewing processes.

The invented process treats beer wort before boiling, degrading gluten and producing peptide fragments that are not perceived by the celiac organism. Then these peptide fragments will be easily removed during the beer wort ripening stage by precipitation and filtration. Another advantage of the invented process is the possibility of recovery of the enzymes employed in the complete gluten protease.

Because enzymes remain fixed to a support or substrate during the gluten degradation process and act at controlled temperature and pH so that they do not denature them, the enzymes may be employed in a very large number of gluten proteases.

Thus, it is possible to obtain a gluten-free beer by the complete protease of this protein, making it possible for people with celiac disease to consume this beer.

Claims

1 - GLUTEN-FREE BEER MANUFACTURING PROCESS, made from grains and raw materials containing this protein, characterized by:

A - for gluten degradation to occur through complete protease of this protein through the use of pepsin (EC 3.4.23.1 and EC 3.4.23.2), trypsin (EC 3.4.21.4), chymotrypsin (EC) enzymes 3.4.21.1), papain (EC 3.4.22.32.33), bromelain (EC 3.4.22.28 to 33), chymosin (EC 3.4.23.4), phycin (EC 3.4.22.3), EC family enzymes 3.4, with emphasis on the enzymes of the mucopepsin subclasses (EC 3.4.23.23), carboxypeptidase A (EC 3.4.17.1), carboxypeptidase B (EC 3.4.17.2) or glyoxa carboxypeptidase (EC 3.4.17.4);

B - for the possibility of reusing the enzymes employed in the complete gluten protease;

C - by removing peptide fragments resulting from gluten degradation;

D - For the fermentation of beer employ yeasts multiplied in gluten free medium to avoid cross or residual contamination.

Gluten-free brewing process according to claim 1, characterized in that enzymes during protease disrupt or hydrolyze gluten peptide bonds, resulting in degradation and formation of peptide fragments that are not recognized by the celiac organism. .

GLUTEN-FREE BEER MANUFACTURING PROCESS, according to claim 1, characterized by the possibility of employing enzymes of one class or one subclass or the combination of enzymes of two or more classes or subclasses depending on the grains used and the characteristics of the process. production of a particular beer, such as temperature, pH, process time and chemical agents added to the drink (antioxidants, preservatives, acidulants, stabilizers, emulsifiers, among others).

4. A GLUTEN-FREE BEER MANUFACTURING PROCESS according to claim 1, characterized in that any type of grain containing gluten is used as raw material, especially wheat, barley, rye, oats and their variants or sub- -species, no matter if they are malted or not.

Gluten-free brewing process according to claim 1, characterized in that the enzymes, during the process of degradation of gluten in the treatment tank, remain adhered to a support or substrate, which will enable their reuse in other treatments.

Gluten-free brewing process according to claim 5, characterized in that the substrate or substrate is chosen from calcium alginate, chitosan, acid cellulose fiber, gelatin, aga, polyacrylamide, chitin and alumina. .

Gluten-free brewing process according to claim 1, characterized in that an amount of one to one hundred containers containing the support or substrate plus the enzymes is contained within a treatment tank.

Gluten-free brewing process according to claim 1, characterized in that the gluten protease time lasts between 30 minutes and 10 hours, and preferably between 4.0 and 6.5 hours.

A GLUTEN-FREE BEER MANUFACTURING PROCESS according to claim 1, characterized in that the process temperature ranges from 10 ° C to 90 ° C, preferably indicating that the protease occurs at temperatures between 40 ° C and 60 ° C. A GLUTEN-FREE BEER MANUFACTURING PROCESS according to claim 1, characterized in that the pH of the treatment ranges from 3.0 to 10.0, preferably from 5.5 to 6.5.

Gluten-free brewing process according to claim 1, characterized in that the process of gluten degradation results in levels of gliadin, hordein, secaline and / or avenin less than 20 ppm, preferably less than 2 ppm, measured values. by the Elisa test.

Gluten-free beer brewing process according to claim 1, characterized in that the removal of peptide fragments resulting from gluten degradation occurs during the beer wort maturation step by precipitation and filtration.

Gluten-free brewing process according to claim 1, characterized in that it uses gluten-free yeasts which have been multiplied in glucose medium or in a medium previously treated by the complete protease, which will not contain gluten or gluten traces.

Gluten-free beer brewing process according to claim 1, characterized in that the gluten degradation process is applicable in the production of alcoholic and non-alcoholic beers, beer bases, beer concentrates and beer additives.