Method to produce maltobionat

FIELD: biotechnologies.

SUBSTANCE: method to produce maltobionat provides for production of a substrate applied in process of production of mash or mesh and containing maltose; and conversion of maltose into maltobionat by means of a reaction catalysed with carbohydratoxidase, for instance, carbohydratoxidase from Microdochium nivale CBS 100236. At the same time maltobionat is formed at the stage of mashing in process of beer production.

EFFECT: invention provides for practically full conversion of maltose in a substrate into a maltobionat.

6 cl, 1 tbl, 3 ex

 

The present invention relates to a method of preserving food and feed products with anti-oxidants derived directly from starch or maltose, are already present in the food product, when using an enzymatic process. Also the present invention relates to a method for multibionta of the starch contained in the products.

Prevention of oxidative destruction of the food or feed is very important for preserving the quality of products. The oxidation products can cause changes in color, taste, aroma, or other unacceptable organoleptic changes. Additionally, oxidation can cause damage to essential amino acids and result in the loss of vitamins. In particular, foods containing polyunsaturated fatty acids are prone to oxidation, which can potentially lead to rancid food.

The oxidation reaction occurs when a molecule of the food product, for example, fatty acids, combines with oxygen in the presence of free radicals; the trace metals, such as metal trace elements, Fe and Cu; or active forms of oxygen such as atomic oxygen, peroxides or hydroxides. Antioxidants are used to suppress these reactions. Examples usually COI is lsemaj antioxidants include butylhydroxyanisole (BHA) and equivalent (BHT), which are widely used in food products high in fats and oils, along with sulfites, which are used primarily as antioxidants to prevent or reduce discoloration of fruits and vegetables. However, it is assumed that BHA and BHT cause tumors when used in high concentrations and, therefore, are not safe for human health, and know that sulfites destroy vitamin B. For these reasons, it is generally preferable biological or natural antioxidants, such as tocopherol (Vitamin E), L-ascorbic acid, citric acid, melanoidin, flavonoids and Gallic acid. To solve the oxidation of the use of chelating agents such as EDTA, siderophores (chelating iron agents from micro-organisms), citric acid and lactobionic acid, due to their ability to prevent the oxidation of metallic trace elements.

In U.S. patent No. 3899604 retrieves maltobiose acid from maltose enzymatic oxidation using Pseudomonas gravlolens species and application maltobiose acid as a food additive; multibionta acid has a slight sour taste and also contributes to the viscosity of food products in which it is contained. Additionally multibionta acid may enhance genuine cowhide leather-makes the th smell and taste of certain foods (improver of taste and smell), as described in U.S. patent No. 3829583. However, there is no indication that multibionta has an antioxidant effect on the food product.

In European patent No. 0384534 B1 describes obtaining maltobiose acid from maltose enzymatic oxidation using a strain of Pseudomonas cepacia. Oxidation occurs not only during long-term storage, but can also occur in the process of obtaining the product, in particular when in the process of obtaining oxygen is present. Therefore, continues to exist the need to obtain natural antioxidants for food.

The present invention relates to a method for preventing oxidative reactions in food and feed products by obtaining multibionta from starch or maltose present in food or feed product, when using an enzymatic process.

According to the present invention oxidative reactions in food and feed products can be prevented or delayed in the process of obtaining multibionta. As far as we know, multibionta first used as antioxidant feed or food product in the process of getting it or after it.

Additionally, the present invention relates to a method for multibionta of the starch component of food is the first or feed product, where it acts as an antioxidant. Therefore, the antioxidant of the present invention can be obtained directly from the product components, thus ensuring 100% natural antioxidant that eliminates separate production and adding antioxidant.

Definitions:

Used in this description, the term "additive" refers to a piece of ground, which is not barley malt. The additive may include any plant material rich in starch, for example, neoslojnennoe grain milling simple, such as barley, rice, corn, wheat, rye, sorghum and easy fermentary sugar and/or syrup.

Used in this description, the term "fraction isolated from food or feed product in the process of getting it" refers to the highlighted part, which essentially contains all the ingredients used in the norm in that part of the process where it is allocated. Preferably the fraction has a high starch content compared to those present in the norm in that part of the process where it is allocated. The fraction can be obtained at any stage of the process to obtain and also may be a finished product. As it turned out, when it is desirable increased starch content in the selected fraction can be added ingredient of the method that contains more starch, or the East starch.

Used in this description, the term "grinding" refers, as to raw materials containing starch and raw materials containing sugar, which is the basis for the beer, for example, barley malt and Supplement.

Used in this description, the term "isolated enzyme" refers to a polypeptide described with enzymatic activity, where the polypeptide is at least 20% pure, preferably at least 40% pure, more preferably at least 60% pure, even more preferably at least 80% pure, and most preferably at least 95% pure, as determined using SDS-PAGE.

Used in this description, the term "malt" refers to any solarenemy grain, particularly barley.

Used in this description, the term "multibionta" refers to maltobiose acid (CAS Reg. No. 534-42-9; (4-O-alpha-D-glyukopiranozil-D-gluconic acid (4-O-alpha-D-Glucopyranosyl-D-gluconic acid)or its salts. Suitable salts include, without limitation Na-multibionta, Ca-multibionta, NH4-multibionta and K-multibionta.

Used in this description, the term "batch" refers to a suspension, in which the starch contained in the liquid mixture comprising grinding in water.

Used in this description, the term "pure maltose" refers to the composition, the cat heaven only contains maltose, water, inorganic salts and potentially sauterelle agent, such as inorganic salts (e.g., a phosphate salt, a carbonate salt, hydrocool and the like), organic salts (stratoforte, sodium acetate and the like) and other organic buffers (such as HEPES, Tris, and the like).

Used in this description, the term "weak", the opposite of "strong" refers to the ability of reason to dissociate. In the context of the present invention a weak base is defined as the base index pKb of at least a 3.5 (for reasons that are able to bind two protons, such as CO32-this index pKb refers to the first stage).

Used in this description, the term "mash" refers to non-fermented liquid obtained after extraction of grinding in the preparation of the batch.

The enzymes.

Multibionta can be obtained by oxidation of maltose. The oxidation can be carried out using bromide, but in the process of getting food is not desirable.

In the present invention multibionta derived from maltose is the product of the enzymatic reaction, in which the oxidoreductase has a substrate specificity to maltose and catalyzes the transformation. The oxidoreductase are enzymes, rolled yousie the transfer of electrons from one molecule to another. Dehydrogenases and oxidases belong to the class of oxidoreductase enzymes. Usually, dihydroorotase require the presence of a cofactor such as NAD/NADP, or coenzyme flavina, such as FAD or FMN, and this can also be attributed to the oxidase. Unless otherwise stated, the enzymes described below and described in the description, are selected enzymes in the presence of the cofactor, if required.

One of the categories oxidoreductase, suitable for use in the present invention, are oxidase that catalyzes the oxidation reaction/recovery, where molecular oxygen (O2) acts as an electron acceptor. For these reasons, the oxygen is reduced to water (H2O) or hydrogen peroxide (H2O2). In particular, carbohydrazide catalyzes the conversion of maltose in maltose-Delta-lactone, which immediately decomposes in water to obtain multibionta. In the way stands out the hydrogen peroxide. Schematically, the reaction can be described as:

maltose+O2+H2O→multibionta+H2O2(formula 1)

Specialist in the field of technology to which the present invention is known and available suitable carboxyhydroxymethyl capable of converting maltose into multibionta. Examples of such carboxyhydroxymethyl represent alasoorituse, C is lolitakids (EC 1.1.99.18), pianosounds (EC1.1.3.10) and exotoxins (EC1.1.3.5). For reference EC 1.1.3._, EC 1.2.3._, EC 1.4.3._, and EC 1.5.3._ or similar classes of enzymes, based on the recommendations of the nomenclature Committee of the International Union of biochemists and molecular biologists (Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB)), other examples of suitable carboxyhydroxymethyl can be easily determined by the expert in the field to which the present invention relates.

Preferred carboxyhydroxymethyl is a microbial carboxyhydroxymethyl, in particular, highlighted carboxyhydroxymethyl.

Hexosaminidase (EC1.1.3.5) is carboxyhydroxymethyl able to oxidize some sugars, including glucose, galactose, maltose, cellobiose and lactose. Enzymes belonging to the class of exotoxins, are the preferred enzymes according to the present invention. Exotoxicity produce in nature some species of marine algae. Such species include, for example, found in the family Gigartinaceae, belonging to the order Gigartinales. Examples of the types of exotoxins produced by marine algae belonging to the Gigartinaceae, are Chondrus crispus and lridophycus flacci. Also potential sources of hexokinase suited for use in the present invention, are other types of the Orsk algae of the order Cryptomeniales, including species Euthora cristata. In particular, exotoxicity suitable for use in the present invention, for example, is extracted from red seaweed lridophycus flaccidum (Bean and Hassid, 1956, J Biol Chem 218: 425-436) or extracted from Chondrus crispus, or Euthora cristata, as described in WO 96/40935, which additionally describes the cloning and recombinant expression of exotoxins from Chondrus crispus, described in WO 96/40935 as SEQ ID NO 30 and 31.

Cellobioside (EC 1.1.99.18) is carboxyhydroxymethyl able to oxidize some sugars, including cellobiose, soluble calorigenically, lactose, kilobyte and maltose. Enzymes belonging to the class of cellobioside, are also preferred enzymes according to the present invention. Cellobioside is an extracellular enzyme produced by various woody fungi, such as white-red mold Phanerochaete Chrysosporium, brown rot Coniophora Puteana and wet rot, such as, Monilia sp., Chaetomium, cellulolyticum, Myceliophthora (Sporotrichum) thermophila, Sclerotium rolfsii and Humicola insolens (Schou et al., 1998, Biochemical Journal 330: 565-571).

Other suitable carboxyhydroxymethyl can be obtained, for example, from mitosporic Pyrenomycetes, such as Acremonium, in particular, A. Strictum, deposited as ATCC 34717 or A. strictum T1 (Lin et al., 1991, Biochimica et Biophysica Acta 1 118: 41-47); A. Fusidioides deposited as IFO 6813; or A. Potronii deposited is output as IFO 31 197. In a preferred embodiment of the invention carboxyhydroxymethyl receive from a source described in (Lin et al., 1991, Biochimica et Biophysica Acta 11 18: 41-47) along with described in JP5084074. In another preferred embodiment of the invention carboxyhydroxymethyl derived from fungi belonging to the genus Microdochium, more preferably fungi are Microdochium nivale and even more preferably fungi are Microdochium nivale, deposited as CBS 100236. Oxidase isolated from CBS 100236, described in detail in WO 99/31990 (SEQ ID no: 1 and 2 WO 99/31990 entered into this description by reference in full).

Getting multibionta fermentation by bacteria of the genus Pseudomonas grown on the substrate containing maltose as described previously (USA 2496297, USA 3862005, USA 3899604 and EP384534). Also the present invention can be used dehydrogenase. Such dehydrogenase enzyme system can be separated from Psedomonas, in particular of P. ovalis, P. schuylkilliensis, P. graveolens (for example, deposited as IFO 3460), P. tragi, P. iodinum, P. amyloderamosa (for example, deposited as ATCC 21262) or P. cepacia (for example, deposited as CBS 659.88 or CBS 658.88).

The amount of oxidase/dehydrogenase usually depends on the specific requirements and specific enzyme. Add the amount oxidase is preferably sufficient to obtain the desired degree of conversion of maltose in multibionta for determining the amount of time. Usually, a sufficient amount of added oxidase is from about 1 to about 10,000 OXU per kg of substrate, preferably from about 5 to about 5000 OXU per kg of substrate, and more preferably from about 5 to about 500 OXU per kg of substrate. Specialist in the field of engineering that applies the present invention, it is known the required number of specific enzyme for regulating the conversion of maltose in multibionta.

In the literature oxidase unit (Oxidase Unit (OXU)) in the norm is defined as the amount of enzyme that oxidizes under certain conditions, one micromole of maltose per minute. However, in the examples given in this description OXU determine how one mg of pure oxidase enzyme, as measured by the standards of enzymes.

In an additional aspect, the present invention relates to multibionta received dual enzyme-catalyzed reaction. In the first reaction get maltose from starch components present in the process of obtaining food or feed product, using the amylase enzyme. The second reaction is carried out oxidation of maltose to multibionta, as described above. Two reactions can be carried out simultaneously or sequentially. In a preferred embodiment of the invention the amylase reaction is carried out first with holding her reaction preferentiality in multibionta.

Amylase can hydrolyze starch with obtaining oligosaccharides as the main product, in particular, maltose, how well-known specialist in the field of engineering that applies the present invention. Amylase may be derived from bacteria or fungi, in particular from a strain of Aspergillus, preferably a strain of A. niger or A. oryzae, or a strain of Bacillus. One example is alpha-amylase, for example, from Bacillus amyloliquefaciens, and amyloglucosidase, for example, from A. niger. Commercial products include BAN and AMG (products of Novo Nordisk A/S, Denmark), Grindamyl A 1000 or A 5000 (available from Grindsted Products, Denmark) and Amylase H and Amylase P (products from Gist-Brocades, the Netherlands). Similarly, it can be used beta-amylase or other enzymes decomposing starch, which results in maltose.

In an additional aspect of the present invention add catalase (EC 1.1 1.1.6) to prevent the restriction reaction, conducted by carboxyhydroxymethyl, and to remove unwanted H2O2in byproduct. Catalase is an enzyme that catalyzes the reaction: 2 H2O2→ O2+2 H2O (formula 2).

As described above, carboxyhydroxymethyl oxygen-dependent, but produces hydrogen peroxide. The advantage of adding catalase in the method according to the present invention is that carboxylato sidasa provided with oxygen and at the same time removes the hydrogen peroxide, obladayuschyy very strong oxidizing properties. This is very important when multibionta receive, as an integrated part of the process of obtaining food or feed product. Suitable catalase well-known specialist in the field of technology to which the present invention is, for example, commercially available catalase Catazyme®from Novozymes A/S.

The receipt.

Getting multibionta fermentation, for example, using bacteria of the genus Pseudomonas grown on the substrate containing maltose, well known from the prior art (U.S. 2496297, USA 3862005, USA 3899604 and EP 384534). Additionally, in WO 99/31990 describes the oxidation of pure maltose to multibionta using carboxyhydroxymethyl.

In one aspect the present invention relates to the production of multibionta processing of starch and/or maltose present initially, upon receipt of a food or feed product, a reaction that is separate from the actual process of obtaining food or feed product. The method of obtaining multibionta includes the following stages:

i) obtaining a substrate containing starch and/or maltose, are applicable in the process of obtaining food and/or feed product;

ii) the conversion of starch into maltose using the enzymatic reaction; and

iii) accepts Amenia maltose in multibionta when using the enzymatic reaction.

The fraction containing starch, can be purified to increase starch content before conversion into maltose. The enzymatic reaction stage ii) is preferably catalyzed by amylase. When the substrate stage i) contains maltose, stage ii) may not be conducted. The enzymatic reaction stage iii) catalyzed by the oxidoreductase/dehydrogenase, preferably one carboxyhydroxymethyl above, still more preferably one of hexokinase above, and most preferably by carboxyhydroxymethyl derived from Microdochium nivale, deposited as CBS 100236. Stage ii and stage iii) may be carried out as single-stage or two-stage process, in which amylase and carboxypeptidase can be added to the reaction mixture, both collectively and amylase can react with the starch in a separate stage before adding carboxyhydroxymethyl in the reaction mixture. The described method allows to obtain an almost complete conversion of the starch/maltose in multibionta, preferably 80%, more preferably 85%, even more preferably 90%, and even more preferably 95%, most preferably 99%, and even most preferably 100% of starch and/or maltose in the substrate becomes multibionta. Multibionta can be added back to the feed is whether the food product in the desired quantity. Optional multibionta obtained by the method as described above can be purified, when its purity is insufficient.

The advantage of this method is the use of components used in the teaching of food or feed product, therefore, multibionta not receive any supplements. Preferably the fraction containing starch and/or maltose obtained from food or feed product comprises from 5% to 60%, more preferably from 10% to 40%, even more preferably from 15% to 30%, even more preferably from 20% to 25% starch and/or maltose. An additional advantage of the method according to the present invention is that other components of the food or feed product does not complicate the process.

The burdening process other components of the food or feed product, for example, can lead to the formation of foam, since the reaction with carboxyhydroxymethyl may require the addition of oxygen in the reaction mixture, which can cause foam containing protein in the reaction mixtures. The conditions for the transformation of starch into maltose using amylase is well known from the prior art. If required, a specialist in the field of technology to which the present invention may choose conditions compatible with conditions glarefree maltose in multibionta, as is described below.

A substrate comprising starch and/or maltose can be obtained, for example, in the process of getting food or feed product for use in the process of irrorata, barley, starch, cassava, corn, maize, millet, oats, potato, rice, rye, sago, soy, sorghum, sweet potatoes and/or wheat. Processes for the production of food products that use raw materials, initially containing starch and/or maltose, include, for example, the brewing, some wines or spirits, the manufacture of soft drinks manufacture of bakery products manufacture of chips or skakovyh food. In particular, when brewing maltose is present initially, because in the mashing process, it is produced by the fermentation. Namely, the high content of maltose is a must. The substrate containing starch and/or maltose used in the method according to the present invention can be a pure starch contained in the feedstock, as described above, preferably such feedstock is crushed, for example, razdroblennoj or crushed and suspended in water. Alternatively, the fraction can be obtained with the process of getting food or feed product, such as mix, mash, snekovogo product, potato chips and soft drink. PR is doctitle substrate, containing starch and/or maltose used in the methods of the present invention, does not contain pure maltose.

The method of obtaining multibionta should be conducted under conditions that allow carboxyhydroxymethyl to convert maltose into multibionta. Such conditions include, without limitation, temperature, pH, oxygen, quantity and characteristics of carboxyhydroxymethyl, other additives, such as, for example, catalase, and reaction time/shutter speed.

A suitable dwell time provides the desired degree of conversion of maltose in multibionta. Typically, a suitable exposure time is chosen from ½ hour to 3 days, preferably from 2 hours to 48 hours, more preferably from 5 hours to 24 hours, most preferably from 8 hours to 18 hours.

Oxygen is an important factor for the method of the present invention, since the conversion of maltose in multibionta is oxygen consumption (see equation 1 above). Therefore, if the oxygen control during the first enzymatic reaction, we usually see a decrease in the initial amount of oxygen that is, if, for example, provide continuous access of air, when the enzymatic reaction has ended, will return to approximately the initial level. When the oxygen level returns more than 90% of the initial level of the enzyme is active reaction ends or at least slowed down considerably, indicating that the entire substrate (e.g., starch, dextrin and/or maltose) turned into multibionta. Therefore, a suitable exposure time may preferably be such period of time that at least lasts as long as the oxygen level in the processed batch will be more than 90% of the initial level, if in fact it is desirable maximum conversion of maltose. Alternatively, the reaction can be controlled by the amount of base required to maintain a constant pH. When the amount of base required to maintain the pH decreases, this indicates that the reaction ends, or at least slowed down considerably. However, the slowdown of the enzymatic reactions can occur not only due to the depletion of the substrate. The stability of the enzyme over time is also a parameter that may have an impact on the reaction. Therefore, if the enzyme is destroyed, it can also cause slow reactions. In this case, the substrate will not lead to a re-increase oxygen and pH.

Suitable sources of oxygen include air (20% oxygen), air enriched with oxygen (the oxygen content of >20%), and net Ki is oxygen. The implementation of the method under a pressure higher than 1 atmosphere, increases the solubility of oxygen and may be preferable when applying. The supply of oxygen in the process may be carried out, for example, continuous mixing of the air with the reaction mixture during the aging.

Alternatively, to ensure the O2enter H2O2in the presence of catalase (see equation 2 above). In the alternative, can be used H2O2originally produced by carboxyhydroxymethyl. The use of H2O2as the oxygen source may be essentially preferred when the method is carried out with the use of immobilized enzymes, when adding oxygen is very difficult or when the foam is formed, for example, in the reaction mixtures containing protein that creates problems when adding oxygen to the air mixing with the reaction mixture. Catalase may be added at any suitable time, for example, together with carboxyhydroxymethyl or during the reaction, when the level of O2reduced, preferably catalase added to the beginning of the exposure (time=0). The advantage of adding catalase together with carboxyhydroxymethyl is that the required amount of oxygen can be significantly reduced (by up to 50%). Therefore, ensure the increase of oxygen, for example, in the form of air, can be significantly reduced. In fact, adding an adequate amount of catalase together with H2O2you can completely cancel additional supply of oxygen. This optional add H2O2can be available from a commercial source.

Therefore, in a preferred embodiment of the invention, essentially all required for oxidation of maltose to multibionta oxygen is produced by adding catalase, which allocates the required oxygen conversion available H2O2. If the number of H2O2the method is limited, there may be added additional H2O2.

Used in this description, the term "essentially all the oxygen is used to describe the oxygen necessary for the adequate completion of the enzymatic reaction, and essentially, there is no need for adding additional oxygen to the intensification process.

In a preferred embodiment of the invention catalase added to the amount that reduces the concentration of H2O2in comparison with those in similar ways without catalase. More preferably the amount of catalase added in the method according to the present invention, it is enough to decrease the number of H2O2at least 25%, 50%,75%, 85% or 95% compared to the comparative control method, where the relative difference is only that do not add catalase, even more preferably the amount of catalase added in the method according to the present invention, as mentioned above, amounts to this, which is enough to achieve 100% reduction in the number of H2O2in comparison with the comparative control method, where the relative difference is only that do not add catalase. Preferably catalase added to the number, which also improves the degree of conversion of maltose in multibionta.

Usually holding temperature dependent carboxyhydroxymethyl and is usually selected according to the optimum reaction temperature for carboxyhydroxymethyl. However, because the increase of temperature decreases the solubility of oxygen, to obtain the optimal process should take into account other factors. Specialist in the field of engineering that applies the present invention, it is known how to balance the optimum temperature in relation to, for example, enzymatic activity and solubility of oxygen. Typically, a suitable temperature is from 0°C to 99°C, more preferably from 5°C to 90°C, more preferably from 15°C to 85°C, even more preferably from 25°C to 80°, most preferably from 30°C to 60°C.

The optimum pH may vary depending on carboxyhydroxymethyl. However, kinetic analysis of carboxyhydroxymethyl from Microdochium nivale (Nordkvist et al., 2007, Biotechnol Bioeng 97: 694-707) indicates that the use of strong bases (NaOH) can have a negative impact on the stability of carboxyhydroxymethyl. Additionally, in WO 97/004082 describes that the increased output lactobionate when using carboxyhydroxymethyl can be obtained when carrying out the method with a stable pH. Therefore, to improve yield multibionta in the method according to the present invention it may be desirable to maintain a stable pH level in the conversion of maltose in multibionta (stage iii, above), adequate addition of the base. In specific embodiments of the invention a stable pH support from about 3.0 to about 9.0 by the addition of base. To maintain the pH within the above limits can be used any reason. In principle, the method can be applied to any substance capable of neutralizing acid selected. Specialist in the field of engineering that applies the present invention, there are many reasons that can be applied in the method according to the present invention, for example, strong bases such as Ca(OH)2, KOH, NaOH, Mg(OH)2 . In a preferred embodiment of the invention to maintain the pH at a stable level using weak base or carbonate. Examples of weak bases include, without limitation CaCO3, Na2CO3, K2CO3, (NH4)2CO3and NH4OH. In the currently preferred weak bases are NH4OH and Na2CO3.

The preferred pH stable for a particular method can be determined by a specialist in the field of engineering that applies the present invention, and depends on many factors. For example, if the food product is a beer, it is known that the pH of the wort is from about 5.0 to 5.7, preferably from about 5.1 to 5.3. Therefore, it is preferable to maintain a stable pH of about 5.3, i.e. in the range from 5.0 to 5.6. The preferred pH for other food/feed product may range from 3.0 to 4.0, for example, juice or soft drinks such as Cola, or from 4.0 to 5.0, for example, for a beer or mayonnaise or dressings; or from 5.6 to 6.5, for example, for meat products; or between 6.6 to 7.5 for dairy and egg products.

It should be understood that the pH multibionta product or composition, including multibionta of the present invention, can also be adjusted to the preferred pH after or at the end of f rotating transformation, for example, when reaching 95% of the desired conversion of maltose can be carried out by lowering the pH to the desired level.

Used in this description, the term "stable pH" includes in the scope of the monitoring and maintaining the pH during the process, in particular within or close when/to a specific index by adding a base. Control and management/maintenance of the pH during the enzymatic process is a standard procedure that can be performed with very high accuracy. Therefore, a stable pH may be the measure supported at a constant level, with a variation of less than 1.5 pH unit, preferably less than 1.0 pH unit, more preferably less than 0.5 pH unit, more preferably less than 0.3 pH units, even more preferably less than 0.2 or 0.1 unit of pH. From this it follows that the limits can be defined for a specific enzymatic process of the present invention, and that pH can be controlled and maintained with the above degree of accuracy in this range. In the method according to the present invention is particular suitable pH range, or a specific pH selected from the range from about pH 3 to about pH 9.

It is preferable to maintain the pH at a stable level, as mentioned above, since the enzymatic reaction is AI. In other words, immediately after adding oxidase in a product containing maltose, to maintain a stable pH add the base, as shown above.

In particular, in the case where it is desirable maximum conversion of maltose, pH is maintained at a stable level, as mentioned above, during the period of time that at least lasts as long as the oxygen level in the reaction mixture will comprise more than about 90% of the initial level, or the amount of base used to maintain a constant pH, corresponds to the desired level of transformation.

Preferably the pH is maintained at a stable level, as mentioned above, during the period of time from 30 minutes to 3 days, preferably from 2 hours to 48 hours, more preferably from 5 hours to 24 hours, most preferably from 8 hours to 18 hours.

In a specific embodiment of the invention the conversion of maltose in multibionta carried out in batches of wort obtained from the stage of mashing. Wort containing multibionta, can be used as an ingredient for parties wort to get beer. In another specific embodiment of the invention the conversion of starch into maltose in multibionta spend lots of dough at the stage of mashing. The method can include adding amylase together with carboxyhydroxymethyl. Batch, who aderrasi multibionta, can be used as an ingredient in the stages of mashing.

A particular aspect of the present invention relates to a method of enzymatic conversion of maltose in multibionta obtaining increased output and/or reduced reaction time. The method is characterized by the following stages:

i) adding carboxyhydroxymethyl in the substrate, including maltose;

ii) exposure of the substrate under conditions that allow carboxyhydroxymethyl to convert maltose into multibionta; and

iii) maintaining the pH value at stage ii) from about 3.0 to about 9.0 by the addition of the base.

In a particular aspect, the substrate used to obtain multibionta may contain pure maltose. Preferably the substrate is produced by conversion of the starch, for example, from the stage of obtaining food or feed product, maltose enzymatic reaction using, for example, amylase, as mentioned above.

Obviously, the methods of the present invention can be used for industrial production of multibionta per se. However, the method can also be part of the process of obtaining food or feed product, in the process of obtaining which initially is maltose.

In another aspect, the present invention relates to the production of multibionta directly (in situ) in the process of getting pexeva is about or feed product processing starch and/or maltose, which was originally present in the process. Therefore, in this aspect multibionta get in the process of getting food or feed product, without conducting a separate stage for the reaction, as mentioned above. The method of obtaining multibionta, when the method is integrated in the process of obtaining food or feed product, includes the following stages:

i) adding an oxidoreductase or dehydrogenases, preferably of carboxyhydroxymethyl in the process of getting food or feed product;

ii) maintaining process under conditions that allow the enzymatic conversion of maltose in multibionta;

iii) the continuation of the process of obtaining food or feed product.

The enzymatic reaction stage (i) can be carried out by reaction of the destruction of the starch, for example, catalyzed by amylase, which can be carried out as endogenous (for example, already in the process of malt)and exogenous (e.g., added before or together with the enzyme at stage i). Carboxyhydroxymethyl and conditions of the enzymatic transformations are essentially the same as described above. However, with respect to the optimal temperature should be taken into account that the reaction takes place in the process of obtaining food/feed product. Therefore, the advantage is that the carb is cigerattes can operate at the optimum for such a process temperatures. The advantage of obtaining multibionta directly in the process of getting food or feed product is that the process is optimized, and there is no need for a separate stage of obtaining multibionta.

In a specific embodiment of the invention multibionta get on stage, get beer, such as stage of mashing or stage of fermentation, by adding at this stage of carboxyhydroxymethyl and potentially catalase. The conversion of maltose in multibionta can be carried out in the batch (grinding+liquid)before mashing process or during the mashing process, or after boiling the wort before fermentation, or even during fermentation. However, later must be present in the food product meets the standard of enzymes. However, for the fermentation of wort into beer must be present in the wort maltose. Therefore, the conversion of maltose in multibionta should be optimized so that maltose is converted into multibionta only partially. Preferably wort contains up to 2% maltose, more preferably up to 5% maltose and most preferably up to 10% maltose.

In the mashing process is usually carried out stepwise controlled temperature rise, where at each stage the action of one enzyme predominates over the effect of the other, the development of the Shay in the proteins, cell walls and starch. The temperature profiles rubbing known from the prior art. In the method according to the present invention the conversion of maltose in multibionta occurs preferably at the stage of saccharification (destruction of starch) from 55°C to 66°C. In a preferred embodiment of the invention carboxyhydroxymethyl active in this temperature limit. Alternatively, the mashing process can be conducted at lower temperatures, long enough to let be the transformation of starch into maltose in multibionta at a temperature of activity carboxyhydroxymethyl. Amylase may be added exogenous at this stage to facilitate the conversion of starch into maltose.

In another embodiment of the invention the receiving multibionta carried out after the fermentation of beer. In this case, the required amount of maltose is provided with carboxyhydroxymethyl and potentially catalase, because all available maltose was converted during fermentation.

In another embodiment of the invention the food product is a snack with high starch content, that is >25%, more preferably 50% or higher and a high content of lipids, that is >10, more preferably above 15%. Amylase, carboxypeptidase and, potentially, catalase can be the ü added to znakowy food product in the process of getting together with traditional ingredients, for example, proteins, such as milk or milk powder, gluten, and soy; eggs (whole eggs, and egg yolk or albumen); shortening, such as granular fat or oil; regenerating agent such as L-cysteine; a sugar; a salt such as sodium chloride, calcium acetate, sodium sulfate or calcium sulfate. Getting multibionta directly in sicovam the food product may to some extent replace the traditional antioxidants, such as ascorbic acid, potassium bromate, potassium Iodate, azodicarbonamide (ADA) or ammonium persulfate. Alternatively, the method can be carried out in accordance with the first aspect of the present invention, where the amylase, carboxypeptidase and potentially catalase is added to the party starchy raw materials used to obtain snekovogo food product. In this case, a high degree of conversion of maltose in multibionta can be guaranteed in such a party, and the party can be added back in the process of getting snekovogo food product.

Cleaning multibionta:

Optional you can clean multibionta any suitable way with getting multibionta product or composition, including multibionta with the desired degree of purity multibionta.

Specialist in the field of technology, to the cat who Roy is the present invention, know how to clean multibionta, and depending on the specific needs of the composition comprises at least 70% multibionta at least 80%, at least 90% multibionta or even at least 95% or at least 99% multibionta.

Suitable cleaning methods multibionta include filtration, ion exchange, concentration and drying.

Composition comprising multibionta, can be used to produce food products such as, for example, a food additive or ingredient of a food product, in particular, as an antioxidant in the food product.

Application multibionta in food and feed products.

Also the present invention relates to the use of multibionta as an antioxidant in the food or feed product, in particular, as a chelating agent.

In one aspect of the present invention multibionta added in an effective amount in the food or feed product. Expert in the technical field to which the present invention can determine the number of multibionta need to obtain antioxidant effects on food or feed product.

Multibionta can be provided in the retrieval process, as described above. When in the process of obtaining initially no is there starch or maltose, multibionta can be added in the production process. Alternatively, multibionta can be added to the finished feed or food product.

The purpose of this invention is the addition of multibionta in the food product to provide antioxidant effects, and does not influence the viscosity of the food product and its ability to enhance the natural smell and taste of certain foods (improver of taste and aroma).

EXAMPLES

EXAMPLE 1.

Getting multibionta.

Na-multibionta derived from maltose by oxidation, catalyzed by carboxyhydroxymethyl (M. Nivale CBS 100236, as described in WO99/31990) and catalase (Catazyme 25L, Novozymes, Denmark). The dosage of the enzyme is: carboxyhydroxymethyl 400 mg protein enzyme/kg of maltose, and catalase 6 g/kg of maltose. Maltose was dissolved at a concentration of 10%at a temperature of 38°C. To conduct the reaction using a reactor with a stirrer, containing 3 l of a solution. During the reaction serves atmospheric air 1 l/min and maintain constant pH 6.4, continuously adding a 1M solution of Na2CO3. The total reaction time is 17 hours. Essentially during the reaction all maltose converted into maltobiose acid.

EXAMPLE 2.

Antioxidant effect multibionta measured by analysis of recovery antioxidants jelly is a (Ferric Reducing Antioxidant Power (FRAP) assay).

Briefly, the FRAP analysis carried out as follows: the complex Fe3+-tripyridyltriazine (TPTZ) is restored to Fe2+-TPTZ at low pH. The form of ferrous (Fe2+) painted blue when measured Spectro-photometry at 593 nm. The method is calibrated using a solution of Fe2+with known concentrations. A higher absorbance indicates a higher antioxidant status. The working reagent for analysis of FRAP is obtained from the following components:

Acetate buffer: 3.1 g CH3COONa•3H2O and 16 ml conc. CH3COOH at ≈800 ml of water. Check that the pH was 3.6. Otherwise govern NaOH/CH3COOH. Add water to 1 L.

The TPTZ solution: 10 mmol/l 2,4,6-three(2-pyridyl)-s-triazine (TPTZ) in 40 mmol/l solution of HCI Fe(III): 20 mm Fe(III)Cl3•6H2O.

The working reagent (receive daily): 50 ml acetate buffer+5,0 ml TPTZ+5.0 ml of a solution of Fe(III).

The analysis is performed by adding 50 μl of the sample in 1.5 ml of the working reagent in 2 ml dark Eppendorf tubes, followed by their incubation in thermomixer at 37°C for 30 minutes. The samples contain multibionta in different concentrations, the standard contains Fe2+and ascorbic acid, and a blank sample contains water. The analysis is repeated three times. Determine the absorbance immediately at 593 nm, a higher absorbance is higher antioxidant status (↑Abs→EN is ioxidant ↑). The results are shown in Table 1.

Table 1
Sodium multibionta in FRAP (g/l) Abs at 593 nm
Sodium multibionta (liquid concentrates)
Abs averageThe standard deviation
5%0,2360,021
2,50%0,1700,002
1%0,1120,006
A blank sample0,02570,00058

Multibionta showed antioxidant capacity, restoring Fe(III) and, therefore, changing the absorption due to the complex iron-tripyridyltriazine. The analysis showed obvious dozozawisimy the effect of antioxidants.

EXAMPLE 3.

Antioxidant effect of carboxyhydroxymethyl and/or catalase in the beer wort.

Beer wort is produced from 50 g finely dissolved barley malt in 250 g of water at a temperature of 53°C. Barley malt clobber for 30 minutes at a temperature of 52°C, increasing 1°C/min for 11 mine is, for 30 minutes at a temperature of 63°C, increasing the temperature 1°C/min for 9 minutes, for 30 minutes at a temperature of 72°C, increasing the temperature by 1°C/min for 6 minutes, for 15 minutes at a temperature of 78°C, followed by cooling to 20°C.

Conduct a series of experiments with adding to the wort of carboxyhydroxymethyl and/or catalase, and comparing with a control sample. Enzymes added at the stage of mashing. Carboxyhydroxymethyl dosed according to activity, measured as LOXU, where one LOXU corresponds to 1 mg of protein enzyme. Catalase is dosed according to activity, measured as CIU. 1 CIU represents the amount of enzyme that decomposes 1 µmol H2O2per minute at pH=7.0 and T=25°C.

To measure the antioxidant capacity of carboxyhydroxymethyl and/or catalase in all samples add 1 mm Fe2+. Oxidation was measured by an indirect method using a comprehensive analysis Xylenol orange (XO-analysis).

In this analysis, hydroperoxidase wort oxidizes Fe2+in the complex Xylenol orange to Fe3+which forms a coloured complex with Xylenol orange. The oxidized complex can be measured spectrophotometrically, lower absorbance is higher antioxidant status (↓ Abs→Antioxidant ↑).

XO of the working reagent is obtained from the following the components:

A: 2.5 mm ammonium iron sulfate, the uranyl, 1.0 mm tetranitride salt Xylenol orange (XO) 1250 mm H2SO4.

B: 4,89 mm-bottled hydroxytoluene (BHT) in methanol.

XO of the working reagent: mix 1 part A with 9 parts B. Stable 1 month in the refrigerator if stored in dark glass vessel.

The analysis begins by adding 100 μl of the sample in 900 ál XO of the working reagent. Incubated at room temperature for 30 minutes under stirring. Each sample is centrifuged at 14000 rpm at 20°C for 10 minutes. The absorbance of the supernatant was measured spectrophotometrically at 560 nm.

The absorbance increased in all samples due to oxidation in painted HO complex Fe2+to Fe3+. Samples of wort with 50 LOXU prevent oxidation compared to control (abs=0,410 control and 0,257 for 50 LOXU). The impact of the worse half the dose of 25 LOXU (abs=0,421), but is improved in combination with 600 CIU catalase (abs=0,308). 1200 CIU catalase prevents the formation of a colored complex (abs=0,264) and influencing dose-dependent, with 600 CIU does not differ greatly (abs=0,439) from the control.

1. The method of obtaining multibionta, comprising the following stages:
a) obtaining a substrate that contains maltose, which can be used in the process of obtaining a wort or mash;
b) conversion of maltose in multibionta by p the shares, catalyzed carbohydratecoated,
this multibionta form at the stage of the mash in the brewing process.

2. The method according to claim 1, in which carbohydrated selected from the group consisting of exotoxins, cellobioside, alldataread and pianosounds.

3. The method according to claim 1, in which carbohydrated get from Microdochium nivale, deposited as CBS 100236.

4. The method according to any one of claims 1 to 3, wherein when carrying out the method add catalase.

5. The method according to claim 4, in which the enzymatic reaction stage b) of claim 1 add catalase.

6. The method according to any one of claims 1 to 5, in which stage b) of claim 1 maintain a stable pH by adding a base during this process.



 

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